Inhalable rapamycin formulation for the treatment of pulmonary hypertension

ABSTRACT

The present invention relates to methods and compositions for the treatment and prophylaxis of pulmonary arterial hypertension (PAH) in a human subject in need of such treatment, the methods comprising the pulmonary administration to the subject, preferably via inhalation of a composition comprising rapamycin or a prodrug or derivative thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/516,219, filed on Mar. 31, 2017, now abandoned, which is a nationalstage entry filed under 35 U. S. C. § 371 of International ApplicationNo. PCT/US2015/054550, filed on Oct. 7, 2015, which claims priority toand the benefit of U.S. Provisional Pat. App. Ser. No. 62/060,988 filedOct. 7, 2014 and U.S. Provisional Pat. App. Ser. No. 62/144,145 filed onApr. 7, 2015, the full disclosures of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor pulmonary delivery by inhalation, the composition comprisingrapamycin for the prophylaxis and treatment of pulmonary hypertension.

BACKGROUND OF THE INVENTION

Pulmonary hypertension (PH) is an increase of blood pressure in thepulmonary artery, pulmonary vein, or pulmonary capillaries, togetherknown as the lung vasculature, leading to chest pain, dizziness,fainting, fatigue, leg swelling, light-headedness during exercise,shortness of breath during activity and/or weakness. The symptoms of PHare similar to those of other more common heart and lung problems. Thosedisorders include asthma, pneumonia, chronic obstructive pulmonarydisease (COPD), left heart failure and/or coronary disease, which makethe diagnosis of PH often challenging. The initial cause of PH remainsunknown. Vasoconstriction or tightening of blood vessels connected toand within the lungs are observed in PH. The walls of the smallest bloodvessels thicken and are no longer able to transfer oxygen and carbondioxide normally between the blood and the lungs. This makes it harderfor the heart to pump blood through the lungs. Over time the affectedblood vessels become both stiffer and thicker in a process known asfibrosis. This further increases the blood pressure within the lungs andimpairs blood flow, increases the work load of the heart, and causeshypertrophy of the right ventricle. The heart is less able to pump bloodthrough the lungs resulting in heart failure and death in patients. Thenormal pulmonary arterial pressure in a person living at sea level has amean value of 8-20 mm Hg at rest. PH is present when mean pulmonaryartery pressure exceeds 25 mm Hg at rest.

The World Health Organization (WHO) has classified PH into variousgroups such as: WHO Group 1-pulmonary arterial hypertension (PAH) (e.g.,idiopathic, heritable (BMPR2, ALK1, endogolin, unknown), drug- and toxininduced, PH associated with connective tissue disease, HIV infection,portal hypertension, congenital heart diseases, schistosomiasis orchronic hemolytic anemia (including sickle cell anemia), persistentpulmonary hypertension of the new born), WHO Group I′-Pulmonaryveno-occlusive disease (PVOD) and/or pulmonary capillary hemangiomatosis(PCH), WHO Group II-pulmonary hypertension owing to left heart disease(e.g., systolic or diastolic dysfunction, valvular disease), WHO GroupIII-PH owing to lung disease and/or hypoxia (e.g. chronic obstructivepulmonary disease, interstitial lung disease, other pulmonary diseaseswith mixed restrictive and obstructive pattern, sleep-disorderedbreathing, alveolar hypoventilation disorders, chronic exposure to highaltitude, developmental abnormalities), WHO Group IV-chronicthromboembolic pulmonary hypertension (CTEPH), and/or WHO Group V-PHwith unclear multifactorial mechanisms (e.g. hematologic diseases:myeloproliferative disease, splenectomy, systemic diseases: sarcoidosis,pulmonary Langerhans cell histiocytosis: lymphangioleiomyomatosis,neurofibromatosis, vasculitis, metabolic disorders: glycogen storagedisease, Gaucher disease, thyroid diseases, and others: tumoralobstruction, fibrosing mediastinities, chronic renal failure ondialysis).

Pulmonary hypertension (PH) has no cure. Treatment only relievessymptoms and slows the progress of the disease. Despite considerableadvances in medical therapy for PH, long-term mortality remains high,largely as a result of heart failure and other cardiovascular diseases.

Some types of PH can be treated if the underlying lung disease is known.Currently treatment options available for those suffering from PH ofunknown causes target cellular dysfunction that leads to constriction ofthe vasculature. Therapies such as prostacyclin, prostanoids,phosphodiesterase-5 inhibitors, endothelin receptor antagonists andother vasodilators primarily work by causing dilation of the pulmonaryvessels. For example, prostacyclin given intravenously through acatheter surgically implanted in the skin improves the quality of life,increases survival, and reduces the urgency of lung transplantation.Vasodilators, such as calcium channel blockers, nitric oxide, andprostacyclin, are often helpful for PH associated with scleroderma,chronic liver disease, and HIV infection. In contrast, these drugs havenot been proven effective for people with PH due to an underlying lungdisease. For PAH diuretics, dixogin and oxygen therapy are oftenprescribed. Unfortunately, many patients respond poorly to thesetherapies or stop responding to them over time. The only remainingoption then is a single and/or double lung transplantation, orheart-lung transplantation to treat PH. Although there is some evidencethat available therapies have secondary effects on vascular remodeling,there are currently no therapies that target abnormal cell proliferationin PH.

The molecular mechanism of PH is not known, but it is believed that theendothelial dysfunction results in a decrease in the synthesis ofendothelium-derived vasodilators such as nitric oxide and prostacyclin.Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is apathological feature of all forms of PH that leads to structuralremodeling and occlusion of the pulmonary vessels. Pulmonary vascularremodeling is due to abnormal proliferation and resistance to apoptosisof cells forming the vascular wall, including PA-SMCs, endothelial cellsand pulmonary adventitial fibroblasts. Intracellular signaling pathwaysinvolving the serine/threonine kinase Akt and mammalian target ofrapamycin (mTOR) are known to be critical in cell proliferation andcancer and may also take part in the proliferation of vascular smoothmuscle cells in PH. In PA-SMCs, Akt and mTOR signaling can be activatedby numerous growth factors, as well as physical stimuli, such as shearstress and hypoxia. Thus, the Akt-mTOR signaling pathway is shared byvarious physical and biological stimuli that act on PA-SMCs and caninduce PH.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus (See U.S. Pat. No. 3,929,992). Rapamycin is an inhibitorof mTOR. The immunosuppressive and anti-inflammatory properties ofrapamycin initially indicated its use in the transplantation field andin the treatment of autoimmune diseases. For example, it was shown toprevent the formation of humoral (IgE-like) antibodies in response to analbumin allergic challenge, to inhibit murine T-cell activation, and toprolong survival time of organ grafts in histoincompatable rodents. Inrodent models of autoimmune disease, it suppresses immune-mediatedevents associated with systemic lupus erythematosus, collagen-inducedarthritis, autoimmune type I diabetes, autoimmune myocarditis,experimental allergic encephalomyelitis, graft-versus-host disease, andautoimmune uveoretinitis.

Rapamycin is also referred to by its generic drug name, sirolimus (seefor example, ANDA #201578, by Dr. Reddys Labs Ltd., approved May 28,2013). Sirolimus is FDA approved and marketed in the United States forthe prophylaxis of organ rejection and renal transplantation under thetrade name RAPAMUNE by Wyeth (Pfizer). It is in the form of an oralsolution (1 mg/ml) or tablet (multiple strengths). Wyeth (Pfizer) alsomarkets a derivative by the tradename TORISEL (temsirolimus) for thetreatment of advanced renal cell carcinoma, which is administeredintravenously. Temsirolimus is a water-soluble prodrug of sirolimus.Cordis, a division of Johnson & Johnson, markets a sirolimus-elutingcoronary stent under the tradename CYPHER. In this context, theantiproliferative effects of sirolimus prevent restenosis in coronaryarteries following balloon angioplasty. US 2010/0305150 to Berg et al.(Novartis) describes rapamycin derivatives for treating and preventingneurocutaneous disorders, such as those mediated by TSC includingtuberous sclerosis, as well as those mediated by neurofibromatosis type1 (NF-1). Rapamycin and its derivatives are further described inNishimura, T. et al. (2001) Am. J. Respir. Crit. Care Med. 163:498-502and in U.S. Pat. Nos. 6,384,046 and 6,258,823.

Rapamycin use in its clinically approved context has several knownadverse effects including lung toxicity (the RAPAMUNE label warns thatit is not indicated for lung transplant patients), increased cancerrisk, and diabetes-like symptoms. Rapamycin is associated with theoccurrence of pulmonary toxicity, usually in the form of interstitialpneumonitis, but pulmonary alveolar proteinosis has also beendocumented. See for example, Nocera et al., Sirolimus Therapy in LiverTransplant Patients: An Initial Experience at a Single Center,Transplantation Proceedings (2008), 40(6), 1950-1952; Perez et al.,Interstitial Pneumonitis Associated With Sirolimus in LiverTransplantation: A Case Report, Transplantation Proceedings (2007),39(10), 3498-3499; Hashemi-Sadraei et al., Sirolimus-associated diffusealveolar hemorrhage in a renal transplant recipient on long-termanticoagulation, Clinical Nephrology (2007), 68(4), 238-244; Pedroso etal., Pulmonary alveolar proteinosis—a rare pulmonary toxicity ofsirolimus, Transplant International (2007), 20(3), 291-296. The cause ofrapamycin-induced pulmonary toxicity is not known. It is the lungtoxicity of rapamycin that is of primary concern for its use in thetreatment of PH.

Severe respiratory adverse events have also been associated withsirolimus use as an anti-cancer therapy under chronic administrationresulting in circulating blood concentrations greater than 1 nanogram/mLrange. For example, the lung toxicity of the sirolimus prodrug,temsirolimus, was documented in a 2009 report noting that “interstitiallung disease is a rare side effect of temsirolimus treatment in renalcancer patients”. Aparicio et al., Clinical & Translational Oncology(2009), 11(8), 499-510; Vahid et al., Pulmonary complications of novelantineoplastic agents for solid tumors, Chest (2008) 133:528-538. Inaddition, a 2012 meta-analysis concluded that 10% of cancer patientsadministered temsirolimus or everolimus may experience mild gradetoxicity with a worsening of quality of life and, in some case,interruption of therapy. See lacovelli et al., Incidence and risk ofpulmonary toxicity in patients treated with mTOR inhibitors formalignancy. A meta-analysis of published trials, Acta oncologica (2012),51(7), 873-879. Furthermore, safety pharmacology studies performed inrats with temsirolimus (see Pharm/Tox section of temsirolimus NDA)showed reductions in respiratory rate as well as alveolar macrophageinfiltration and inflammation in the lungs (see Pharmacology Review fortemsirolimus NDA 22088 available from the US FDA website). These adverseeffects were observed under conditions of relatively high concentrationsof the drug in the circulating blood volume as a result of systemicadministration.

Despite its potential for toxicity to the lung, rapamycin has beeninvestigated as a potential treatment for PH in various animal models(Ghofrani et al J. Am. Coll. Cardiol. 54(1):S108-S118 (2009); Paddenberget al BioMed Central 8 (15): 1-12 (2007)). US Patent Application2014/0271871 and WO2008/137148 (collectively, “the Abraxisapplications”) describe methods for treating, stabilizing, preventing,and/or delaying PH by administering nanoparticles of rapamycin and acarrier protein such as albumin. Various modes of administration ofthese rapamycin nanoparticles are generally described includinginhalation. But an aerosol formulation of rapamycin for deliverydirectly to the lungs would be considered highly unlikely to succeed inview of rapamycin's well-known lung toxicity, as exemplified by thearticles cited above. Indeed, the prophetic clinical trial designsdescribed in the Abraxis applications include only intravenousadministration. There is no data in the Abraxis applications that wouldindicate to the skilled person that the problems associated with lungtoxicity of rapamycin have been overcome. A U.S. patent application byLehrer published in 2013 reflects the view that “[r]apamycin (sirolimus)cannot be safely inhaled because of its well-documented lung toxicity,interstitial pneumonitis”. See US 20130004436, citing Chhajed et al.(2006) 73:367-374. The Lehrer patent application is directed tocompositions and methods for treating and preventing lung cancer andlymphangioleiomyomatosis. Although some earlier publications, such asU.S. Pat. No. 5,080,899 to Sturm et al. (filed February 1991) and U.S.Pat. No. 5,635,161 (filed June 1995), contain some generic descriptionof rapamycin for delivery by inhalation, such generic descriptions wereunsupported by any evidence and came before the many reported incidencesof rapamycin-induced lung toxicity that appeared following its morewidespread adoption as an immunosuppressant in the transplantationcontext and as an inhibitor of cellular proliferation in the anti-cancercontext, as evidenced by the reports discussed above.

WO 2011/163600 describes an aerosol formulation of tacrolimus, whichlike rapamycin is a macrolide lactone. But tacrolimus is a distinctchemical entity from sirolimus and the molecular target of tacrolimus iscalcineurin, not mTOR, and unlike rapamycin, tacrolimus did not showlung toxicity and in fact is indicated for preventing rejectionfollowing lung transplantations.

In view of the wide-spread recognition of the potential forrapamycin-induced lung toxicity, a pharmaceutical composition comprisingrapamycin for pulmonary delivery was not considered to be a viabletherapeutic option in humans.

Delivery of drugs to the lung by way of inhalation is an important meansof treating a variety of conditions, including such common localconditions as cystic fibrosis, pneumonia, bronchial asthma and chronicobstructive pulmonary disease, some systemic conditions, includinghormone replacement, pain management, immune deficiency, erythropoiesis,diabetes, lung cancer, etc. See review by Yi et al. J. Aerosol Med.Pulm. Drug Deliv. 23:181-7 (2010). Agents indicated for treatment oflung cancer by inhalation include cisplatin, carboplatin, taxanes, andanthracyclines. See e.g., U.S. Pat. Nos. 6,419,900; 6,419,901;6,451,784; 6,793,912; and U.S. Patent Application Publication Nos. US2003/0059375 and US 2004/0039047. In addition, doxorubicin andtemozolomide administered by inhalation have been suggested for treatinglung metastases. See e.g., U.S. Pat. No. 7,288,243 and U.S. PatentApplication Publication No. US 2008/0008662.

There is a need for pharmaceutical formulations of rapamycin, itsprodrugs, derivatives, and analogues, that can safely be delivereddirectly to the lungs, preferably by inhalation, in order to provide amore effective dosage form for the treatment and prophylaxis of diseasesand disorders affected by the TOR signaling pathway that reduces oreliminates the toxicities and adverse events associated with oral dosageforms of rapamycin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B: LC-MS/MS Chromatogram of 10.6 ng/mL Rapamycin (A) andInternal Standard Triamcinolone (IS) (B) in Mouse Blood.

FIG. 2A-B: Representative Chromatograms of 10.6 ng/mL Rapamycin (A) andInternal Standard Triamcinolone (IS) (B) in Mouse Lung Homogenate.

FIG. 3: Calibration Curve for Rapamycin in Mouse Blood.

FIG. 4: Calibration Curve for Rapamycin in Mouse Lung Homogenate.

FIG. 5A-B: Representative Chromatograms of Rapamycin (A) and InternalStandard Triamcinolone (IS) (B) in Blood from Mouse 2-07 AdministeredRapamycin by OPA.

FIG. 6A-B: Representative Chromatograms of Rapamycin (A) and InternalStandard Triamcinolone (IS) (B) in Lung Homogenate from Mouse 2-07Administered Rapamycin by OPA.

FIG. 7A-B: Rapamycin inhibits S6 phosphorylation (A) and the growth ofTSC2 mutant cells (B).

FIG. 8A-B: S6 Phosphorylation in Mouse Lung Following (A) OPA and oraladministration of rapamycin, and (B) administration via inhalation witha target dose of 0.354 mg/kg.

FIG. 9: Predicted rapamycin blood concentrations for pulmonaryadministration repeated once daily.

SUMMARY OF THE INVENTION

The present invention is based, in part, upon the development of a safeand effective aerosol formulation of a rapamycin composition that iscapable of delivering amounts of the rapamycin composition to targettissues effective to exert potent biological activity in those targettissues while minimizing rapamycin associated toxicity. In addition, theinvention exploits the discovery of the surprising pharmacokinetics of arapamycin composition formulated as described herein. As discussed inmore detail infra, the rapamycin composition delivered directly to thelungs produced markedly higher concentrations of drug in the lungtissue. The amount of drug in lung tissue was unexpectedly higher thanwhat was predictable from previous oral and intravenous studies. Andsurprisingly, even relatively high amounts of rapamycin delivereddirectly to the lungs did not result in toxicity to the lung tissue.Moreover, the amount of drug in lung tissue achieved by the methodsdescribed here is demonstrated to be effective to exert potentbiological activities including inhibition of cell growth and viabilityas well as inhibition of S6 phosphorylation in the target tissue. Thesebiological activities indicate that the delivered dose of the rapamycincomposition according to the claimed methods is sufficient to inhibitmTOR signaling in the target tissues. Thus, these results demonstratethat the aerosol formulations described here are capable of delivering alow, yet therapeutically effective, dose of rapamycin providing for verylow systemic exposure to the drug combined with high efficacy. Theresult is a markedly improved therapeutic index for rapamycin whenadministered according to the present invention.

The present invention provides pharmaceutical aerosol formulations of arapamycin composition for delivery directly to the lungs. In thiscontext, the term “aerosol formulation” may refer to an aqueouscomposition, a dry powder composition, or a propellant-basedcomposition, as described in more detail infra. An aerosol formulationof the invention may be delivered to a subject in different ways, forexample nasally or perorally, e.g., by inhalation. As used herein, theterm “rapamycin composition” may refer to rapamycin itself, preferablyin the amorphous form described as sirolimus, or a prodrug, orderivative thereof. In one embodiment, a rapamycin composition of theinvention provides an amount of rapamycin effective to inhibit mTORsignaling in a target tissue with low or no toxicity to the tissue, andconcomitant blood levels of rapamycin that are less than about 1 ng/ml.

The compositions of the invention are expected to present an improvedsafety profile, especially with respect to their chronic or prolongeduse, compared to current dosage forms of rapamycin which result inpersistent blood concentrations in the range of 1 ng/mL to 15 ng/mL.This is expected due to the low pulmonary toxicity of the presentcompositions combined with the probability of no or substantially feweradverse events due to systemic exposure to rapamycin in view of the lowblood levels of rapamycin produced. Accordingly, the compositions of theinvention are expected to demonstrate a greater therapeutic indexcompared to existing dosage forms delivered via the gastrointestinaltract or intravenously.

In one embodiment, a rapamycin composition of the invention provides animproved safety profile, as evidenced by a higher therapeutic index,especially with respect to its chronic or prolonged use, compared toother dosage forms of rapamycin, for example oral or intravenous dosageforms.

The present invention is directed to compositions and methods for thetreatment and prophylaxis of pulmonary arterial hypertension (PAH) usinga pharmaceutical composition comprising rapamycin designed for pulmonarydelivery, preferably by inhalation. In one embodiment, the inventionprovides a method for the treatment and prophylaxis of PAH in a humansubject in need of such treatment, the method comprising administeringto the subject a pharmaceutical aerosol formulation comprising arapamycin composition, as described herein. In one embodiment, thepresent invention provides compositions and methods for the treatmentand prophylaxis of PAH by administering once daily to a human subject inneed thereof a pharmaceutical aerosol formulation comprising a rapamycincomposition in an amount effective to achieve a maximum blood level ofthe rapamycin composition of from about 0.25 to 0.75 ng/ml and a bloodtrough level of from about 0.075 to 0.25 ng/ml. In one embodiment, themaximum blood level is about 0.5 ng/ml, the blood trough level is about0.1 ng/ml, and the amount of the rapamycin composition in theformulation is from about 25 to 100 micrograms, or about 50 micrograms,delivered once a day.

The aerosol formulations of the invention may be formulated with arapamycin composition alone, or in combination with one or moreadditional therapeutic agents, in the same dosage form. In addition, theaerosol formulations of the invention may be administered alone, or incombination with one or more additional therapies, each administeredeither by the same or a different route, e.g., orally, intravenously,etc. In one embodiment, the aerosol formulations of the invention may beadministered in combination with one or more additional therapeuticregimens for the treatment of PAH.

In one embodiment, the present invention provides a pharmaceuticalaerosol formulation comprising a rapamycin composition effective toachieve a therapeutic level of the composition in a target tissue. Inone embodiment, the target tissue is the lung. In one embodiment, thetherapeutic level is determined 12 or 24 hours after delivery,preferably 24 hours after delivery. In one embodiment, the therapeuticlevel is sustained for at least 24 hours after delivery. In oneembodiment, the lung to blood concentration ratio of the composition 24hours after delivery is from about 5 to 50 or from about 10 to 30.

In one embodiment, the amount of the rapamycin composition in theaerosol formulation is from 5 to 500 micrograms, from 10 to 250micrograms, from 15 to 150 micrograms or from 20 to 100 micrograms. Inone embodiment, the amount of the rapamycin composition in the aerosolformulation is 20, 40, 50, 100, 125, or 250 micrograms.

In one embodiment, the rapamycin composition is sirolimus. In oneembodiment, the rapamycin composition is selected from the groupconsisting of everolimus, temsirolimus, ridaforolimus, umirolimus, andzotarolimus.

In one embodiment, the rapamycin composition is sirolimus and has anisomeric B:C ratio of greater than 30:1 or greater than 35:1. In oneembodiment, the rapamycin composition has an isomeric B:C ratio of 3.5%to 10%.

In one embodiment, the step of administering the composition to thesubject produces particles comprising rapamycin having an average meandiameter in the range of 0.1 to 10 microns. In one embodiment, the stepof administering the composition to the subject produces particlescomprising rapamycin having an average mean diameter in the range of 0.5to 6 microns.

In one embodiment, the method further comprises one or more additionaltherapies or therapeutic regimens.

In one embodiment, the aerosol formulation of the invention is adaptedfor once daily administration and, according the methods describedherein, the aerosol formulation is administered once a day.

In one embodiment, the aerosol formulation is a dry powder compositionsuitable for delivery by inhalation. In one embodiment, the dry powdercomprises the rapamycin composition in the form of microparticles (i.e.,microparticulate rapamycin), particles of a carrier, and one or moreoptional excipients. In one embodiment, the microparticles consist ofparticles of drug having mean diameters from about 0.1 to 10 microns orfrom about 1 to 5 microns. In one embodiment, the particles have a meandiameter of about 1.5 to 4 microns, about 1.5 to 3.5 microns, or about 2to 3 microns. The carrier may be selected from the group consisting ofarabinose, glucose, fructose, ribose, mannose, sucrose, trehalose,lactose, maltose, starches, dextran, mannitol, lysine, leucine,isoleucine, dipalmitylphosphatidylcholine, lecithin, polylactic acid,poly (lactic-co-glutamic) acid, and xylitol, and mixtures of any of theforegoing. In one embodiment, the carrier comprises or consists of ablend of two different carriers. The particles of carrier may havediameters ranging from to 200 microns, from 30 to 100 microns, or lessthan 10 microns. Where the carrier consists of a blend of two differentcarriers, each carrier consists of particles of a different size range,measured as average particle diameter. In one embodiment, the carrierconsists of a blend of two different carriers, a first carrier and asecond carrier. The first carrier consists of particles having diametersranging from about 30-100 microns and the second carrier consists ofparticles having diameters of less than 10 microns. The ratio of the twodifferent carriers is in the range of from 3:97 to 97:3. In oneembodiment, the ratio of the two different carriers is in the range offrom 97:3 or from 95-98:2-5. In one embodiment, the carrier consists ofa blend of two different lactose carriers. The drug to carrier ratio inthe powder may be from 0.5% to 2% (w/w). In one embodiment, the drugcarrier ratio in the powder is 1% (w/w).

The amount of the rapamycin composition in the aerosol formulation isfrom about 0.1% to 20% (w/w) based upon total weight of the composition.In one embodiment, the amount is from about 0.25% to 2% (w/w).

In one embodiment, one or more optional excipients is present in thecomposition and is selected from a phospholipid and a metal salt of afatty acid, and mixtures of the foregoing. In one embodiment, thephospholipid is selected from dipalmitylphosphatidylcholine andlecithin. In one embodiment, the metal salt of a fatty acid is magnesiumstearate. In one embodiment, the excipient or excipients is coated onthe carrier particles in a weight ratio of excipient to large carrierparticle ranging from 0.01 to 0.5%.

In one embodiment, the amount of the rapamycin composition in theaerosol formulation is an amount effective to inhibit the biologicalactivity of mTORC1. In one embodiment, the amount is an amount effectiveto inhibit the phosphorylation of the S6K protein.

In one embodiment, the amount the rapamycin composition in the aerosolformulation is an amount effective to achieve a respirable dose of from5 to 500 micrograms delivered to the lung. In one embodiment, therespirable dose is about 5, about 20, about 50, about 100 or about 250micrograms. In one embodiment, the respirable dose is about 20micrograms, to about 50 micrograms, or about 100 micrograms. In oneembodiment, the amount is an amount effective to produce a concentrationof the rapamycin composition in the lung tissue of from 1 ng/g to 1microgram (μg)/g. In one embodiment, the concentration of the rapamycincomposition in the lung tissue is from about 5-30 ng/g. In oneembodiment, the concentration in the lung is about 5 ng/g, about 10ng/g, about 15 ng/g about 20 ng/g, about 25 ng/g, about 30 ng/g, about50 ng/g, about 60 ng/g, about 100 ng/g, or about 200 ng/g. In accordancewith the foregoing embodiments, the concomitant blood through level ofthe rapamycin composition is less than 5 ng/ml, less than 2 ng/ml, lessthan 1 ng/ml, less than 0.5 ng/ml, or less than 0.25 ng/ml.

In one embodiment, the rapamycin composition persists in lung attherapeutic levels of about 1 ng/g, about 5 ng/g, about 10 ng/g, about15 ng/g, about 20 ng/g, about 25 ng/g, about 50 ng/g, or about 100 ng/gfor a period of time after administration, preferably to a humansubject, the period of time selected from about 6 to 10 hours, about 6to 14 hours, about 6 to 24 hours, and about 6 to 72 hours. In oneembodiment, the period of time is selected from about 12 hours, about 14hours, about 24 hours, and about 72 hours.

In one embodiment, the rapamycin composition persists in lung attherapeutic levels of about 5 to 100 ng/g or from about 5 to 30 ng/g fora period of time that is about 12 or 24 hours. In one embodiment, therapamycin composition persists in lung at therapeutic levels of about 5ng/g, about 10 ng/g, about 20 ng/g, about 30 ng/g, about 50 ng/g, about60 ng/g, about 70 ng/g, about 80 ng/g, or about 90 ng/g. In oneembodiment, the rapamycin composition persists in lung at therapeuticlevels of at least 5 ng/g, at least 20 ng/g, or at least 30 ng/g. In oneembodiment, the rapamycin composition persists in lung at therapeuticlevels of from about 20 ng/g to about 30 ng/g or from about 50 ng/g toabout 80 ng/g.

In one embodiment, the formulation has a fine particle fraction (FPF)greater than 20% with a corresponding fine particle dose (FPD) rangingfrom 5 micrograms to 2 milligrams, preferably less than 0.5 milligrams,following 1 to 12 months or 1 to 36 months of storage. In oneembodiment, respirable dose, which is the dose delivered to the lung,also referred to as the delivered dose (DD) or emitted dose (ED), rangesfrom 10 micrograms to 2.5 milligrams, preferably less than 0.5milligrams. In one embodiment, the delivered dose is from about 20 to100 micrograms, from about 10 to 25 micrograms or from about 30 to 60micrograms. In one embodiment, the delivered dose is 20 or 50micrograms. In one embodiment, the delivered dose is 100 micrograms.

In one embodiment, the respirable dose of the rapamycin composition isabout 20 micrograms, the concentration of drug in the lung tissue isfrom about 5 to 25 ng/g, the Cmax in blood is less than 1.0 ng/ml, orfrom about 0.50 ng/ml to 1.0 ng/ml, or about 0.50 ng/ml to 0.90 ng/ml,the blood trough concentration of drug at 24 hrs post-dosing is lessthan about 0.20 ng/ml, and the steady-state concentration of drug in theblood at 14 days post-dosing is less than about 0.90 ng/ml, or less thanabout 0.80 ng/ml.

In one embodiment, the respirable dose of the rapamycin composition isabout 50 micrograms, the concentration of drug in the lung tissue isabout 2 to 15 ng/g, the Cmax in blood is less than 2.0 ng/ml, or fromabout 0.25 ng/ml to 0.1 ng/ml, or about 0.10 ng/ml to 0.5 ng/ml, theblood trough concentration of drug after a single dose, 24 hrspost-dosing is less than about 0.10 ng/ml, and the trough concentrationof drug in the blood after 5 days repeated, once-daily, is less thanabout 1.0 ng/ml, or less than about 0.50 ng/ml. In one embodiment, theformulation is adapted for once daily administration.

In one embodiment, the formulation further comprises one or moreadditional therapeutic agents.

The invention also provides for the use of the compositions of theinvention for the treatment and prophylaxis of PAH in a human subject inneed of such treatment. In one embodiment, the invention provides amethod for treatment and prophylaxis of PAH in a human subject in needof such treatment or prophylaxis, the method comprising administering tothe subject via inhalation a composition or unit dosage form describedherein.

In one embodiment, the composition delivers an amount of drug effectiveto improve the subject's pulmonary function as measured by forced vitalcapacity (FVC) and forced expiratory volume (FEV1).

The invention also provides a unit dosage form comprising an aerosolformulation comprising a rapamycin composition as described herein,wherein the amount of the rapamycin composition is from about 5 to 2500micrograms, from 20 to 500 micrograms, or from 50 to 250 micrograms. Inone embodiment, the amount of the rapamycin composition is from about 50to 125 micrograms. In one embodiment, the amount of the rapamycincomposition is about 40, about 50, about 100, about 125, or about 250micrograms. In one embodiment, the amount of the rapamycin compositionis about 250 micrograms.

In one embodiment, the unit dosage form is a capsule suitable for use ina dry powder inhaler device. In one embodiment, the capsule containsfrom 1 mg to 100 mg of the powder (total amount, including the rapamycincomposition, carrier, and any optional excipients) or from 10 mg or 40mg of the powder. The capsule may be a gelatin, plastic, or cellulosiccapsule, or in the form of a foil/foil or foil/plastic blister suitablefor use in a DPI device.

The invention also provides a pharmaceutical package or kit comprising acomposition or unit dosage form described herein, and instructions foruse.

In one embodiment, the formulation is produced by a wet polishingprocess comprising the steps of preparing an aqueous suspension of drug,subjecting the drug suspension to microfluidization, and spray-dryingthe resulting particles to form a dry powder.

In one embodiment, the rapamycin composition is sirolimus, the carrierconsists of a blend of two different lactose carriers, the first carrierconsists of particles having average diameters ranging from about 30-100microns and the second carrier consists of particles having averagediameters of less than 10 microns, the ratio of the two differentcarriers is about 97:3 to 3:97, and the amount of rapamycin is from 25to 1400 micrograms.

The invention also provides a dry powder delivery device comprising areservoir containing a composition or unit dosage form described herein.The reservoir may be an integral chamber within the device, a capsule,or a blister. In one embodiment, the device is selected from Plastiape®RS01 Model 7, Plastiape® RS00 Model 8, XCaps®, Handihaler®, Flowcaps®TwinCaps®, and Aerolizer®. In one embodiment, the device is selectedfrom Plastiape® RS01 Model 7 or Plastiape® RS00 Model 8. In oneembodiment, the device is Plastiape® RS00 Model 8.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for thetreatment and prophylaxis of PAH in a human subject in need of suchtreatment. The human subject in need of such treatment is one who hasbeen diagnosed with PAH. In one embodiment, the methods compriseadministering to the subject via inhalation a composition comprisingrapamycin in a suitable carrier, and optionally one or more additives.The term “rapamycin” is used generically throughout this inventiondisclosure to refer to rapamycin itself, also referred to as sirolimus,as well as to its prodrugs (such as temsirolimus) and derivatives.Derivatives of rapamycin include compounds that are structurally similarto rapamycin, are in the same chemical class, are rapamycin analogs, orare pharmaceutically acceptable salts of rapamycin or its derivatives.Further description and examples of rapamycin, its prodrugs, andderivatives are provided in the following section.

The compositions described herein are referred to as “aerosolformulations” and are meant to describe aerosolizable compositionssuitable for producing respirable particles or droplets containing arapamycin composition, which as described above refers to rapamycinitself, preferably in the amorphous form described as sirolimus, or aprodrug or derivative thereof. In one embodiment, the rapamycincomposition is selected from sirolimus, everolimus, and temsirolimus. Inone embodiment, the rapamycin composition is sirolimus. The aerosolformulations described herein may comprise the rapamycin composition, acarrier, and optionally one or more additives. The aerosol formulationsmay be in the form of an aqueous solution, a dry powder, or a mixture ofone or more pharmaceutically acceptable propellants and a carrier, asdescribed in detail in the section below entitled “Compositions forInhalation”.

The present invention also provides methods for the treatment andprophylaxis of PAH in a human subject in need of such treatment, themethods comprising the step of pulmonary administration of an aerosolformulation of the invention to the subject. In one embodiment, theadministered dose of the composition is sufficient to achievetherapeutic levels of the rapamycin composition in the lung tissue whilemaintaining a low blood level, or blood trough level, in a subject. Forexample, the therapeutic levels of the rapamycin composition may be fromabout 1 ng/g, about 5 ng/g, about 10 ng/g, about 15 ng/g, about 20 ng/g,about 25 ng/g, about 50 ng/g and the blood trough level is from 0.01 to0.15 ng/ml, from 0.075 to 0.350 ng/ml, from 0.150 to 0.750 ng/ml, from0.750 to 1.5 ng/ml, or from 1.5 to 5 ng/ml. In one embodiment, theadministered dose is sufficient to achieve a therapeutic level of therapamycin composition in the lung of from about 5 ng/g to 50 ng/g, orfrom about 5 ng/g to 20 ng/g and a blood trough level of the rapamycincomposition of less than 5 ng/ml, less than 2 ng/ml, less than 1 ng/ml,or less than 0.5 ng/ml.

Preferably, the aforementioned therapeutic levels are achieved byadministering an aerosol formulation described herein once a day. In oneembodiment, the total daily dose of the rapamycin composition is in therange of from 5 to 100 micrograms, from 20 to 250 micrograms, from 50 to500 micrograms (0.05 to 0.5 milligrams), from 250 to 1000 micrograms(0.25 to 1 milligrams) or from 500 to 2000 micrograms (0.5 to 2milligrams). In one embodiment, the total daily dose is less than 500micrograms, less than 100 micrograms, less than 50 micrograms, less than20 micrograms, or less than 10 microgram. In one embodiment, the totaldaily dose is less than 500 micrograms, less than 250 micrograms, lessthan 100 micrograms, less than 50 micrograms, or less than 10micrograms. In one embodiment, the total daily dose administered to thesubject is less than 0.5 mg or less than 0.25 mg per day. Furtheraspects of pulmonary delivery and dosing, including combinationtherapies, are described in the section below entitled “PulmonaryAdministration and Dosing”.

In one embodiment, the methods of the invention comprise administeringrapamycin via a pulmonary route in combination with one or moreadditional agents. In one embodiment, the one or more additional agentsis selected from a prostanoid, a phosphodiesterase inhibitor, or anendothelin antagonist. In one embodiment, the prostanoid is selectedfrom the group consisting of a prostaglandin, a thromboxane, and aprostacyclin The one or more additional agents may be administered bythe same or a different route of administration as the rapamycincomposition. For example, the agent may be administered by inhalation,intranasally, orally or intravenously.

The methods and compositions of the invention are effective to treat PAHin a subject in need of such treatment, preferably a human subject. Asused herein, the effective amount of a composition of the inventionrefers to the amount sufficient to reduce or ameliorate the progression,severity, and/or duration of PAH or one or more symptoms of PAH, toprevent the advancement of PAH, cause the regression of PAH, to preventthe development or onset of one or more symptoms associated with PAH, orenhance or improve the prophylactic or therapeutic effect(s) of anothertherapy (e.g., a prophylactic or therapeutic agent) with respect to theseverity or onset of one or more symptoms of PAH, or with respect to thedevelopment or progression of PAH. In specific embodiments, with respectto the treatment of PH, a therapeutically effective amount refers to theamount of a therapy (e.g., therapeutic agent) that inhibits or reducesthe proliferation of vascular cells, inhibits or reduces smooth musclecell proliferation, or reduces thickening of intrapulmonary vessels orimproves FVC or FEV1 or reduces the amount of pleural effusiondetectable by radiologic examination. In a preferred embodiment, atherapeutically effective amount of a therapy (e.g., a therapeuticagent) reduces the proliferation of vascular cells or the thickening ofintrapulmonary vessels by at least 5%, preferably at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% relative to a control (e.g., phosphate bufferedsaline (“PBS”)). Thus, in the context of the methods of the invention,the terms “treat”, “treatment”, and “treating” refer to the reduction ofthe severity, duration, or progression of PAH or one or more symptomsassociated with PAH. In specific embodiments, these terms may refer tothe inhibition of proliferation or reduction in proliferation of smoothmuscle cells, the inhibition or reduction in the thickening ofintrapulmonary vessels, the development of progression of pulmonaryvascular remodeling, or the development or progression of cardiovasculardisease such as heart failure or right ventricular wall thickening,which can be detected by echocardiogram or electrocardiogram.

In one embodiment of the methods of the invention, the composition isadministered in a dose effective to improve the subject's pulmonaryfunction as measured by forced vital capacity (FVC) and forcedexpiratory volume (FEV1). In another embodiment, the composition isadministered in a dose effective to monitor improvements in the rightventricular—right atrial pressure gradient and/or the right and leftventricular chamber sizes using echocardiography.

In certain embodiments, the methods include pulmonary administration ofa composition of the invention as the primary therapy. In otherembodiments, the administration of a composition of the invention is anadjuvant therapy. In either case, the methods of the inventioncontemplate the administration of a composition of the invention incombination with one or more additional therapies for the treatment of adisease or disorder. The terms “therapy” and “therapies” refer to anymethod, protocol and/or agent that can be used in the prevention,treatment, management or amelioration of a disease or disorder, or oneor more symptoms thereof.

The one or more additional therapies may be administered prior to (e.g.,5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of acomposition of the invention.

In some embodiments, an additional therapeutic agent is formulated forco-administration with a composition of the invention in a dosage formfor pulmonary administration. In other embodiments, an additionaltherapeutic agent is administered separately from the dosage form thatcontains rapamycin, and by the same or different route of administrationas the rapamycin. The methods of the invention also contemplate acombination of one or more additional therapeutic agents foradministration concomitantly with, before, or after the administrationof the dosage form comprising rapamycin.

In certain embodiments, the methods of the invention are effective tomanage PAH in a subject having PAH. In this context, the terms “manage”,“managing”, and “management” refer to the beneficial effects that asubject derives from a therapy which does not result in a cure. In oneembodiment, PAH is managed in the subject if its progression is slowedor stopped during treatment with rapamycin according to the methods ofthe invention. In another embodiment, PAH is managed in the subject ifone or more symptoms associated with PAH is ameliorated or stabilized(i.e., the symptom does not worsen during the course of treatment).

In some embodiments, the compositions provided herein inhibit and/orreduce abnormal cell proliferation in the pulmonary artery. In someembodiments, the abnormal cell proliferation is abnormal cellproliferation of smooth muscle and endothelial cells. In someembodiments, the compositions provided herein inhibit and/or reduceangiogenesis in the pulmonary artery. In some variations, angiogenesisin the pulmonary artery is inhibited and/or reduced by suppression ofproduction of matrix metalloproteinases. In some embodiments, thecompositions provided herein inhibit and/or reduce inflammation in thepulmonary artery. In some embodiments, inflammation is inhibited and/orreduced by effecting neutrophil and T cell function.

In some embodiments, the composition is an amount sufficient to increasebasal AKT activity, increase AKT phosphorylation, increase PI3-kinaseactivity, increase the length of activation of AKT (e.g., activationinduced by exogenous IGF-1), inhibit serine phosphorylation of IRS-1,inhibit IRS-1 degradation, inhibit or alter CXCR4 subcellularlocalization, inhibit VEGF secretion, decrease expression of cyclin D2,decrease expression of survivin, inhibit IL-6-induced multiple myelomacell growth, inhibit cell proliferation, increase apoptosis, increasecell cycle arrest, increase cleavage of poly(ADPribose) polymerase,increase cleavage of caspase-8/caspase-9, alter or inhibit signaling inthe phosphatidylinositol 3-kinase/AKT/mTOR and/or cyclin D1/retinoblastoma pathways, inhibit angiogenesis, and/or inhibitosteoclast formation.

In one embodiment, the rapamycin is administered in a dose effective toimprove one or more of the following: cardiopulmonary exercise testing,serial invasive hemodynamic testing, functional residual capacity, serumVEGF-D, quality of life and functional performance, 6 minute walkdistance, and diffusing capacity of the lung for carbon monoxide. In oneembodiment, rapamycin delivered via a pulmonary route achieves bloodlevels of rapamycin effective to limit the thickening of pulmonaryvessels in the lungs. In one embodiment, the efficacy of theadministered dose of rapamycin is measured by any one or more of theforegoing.

In one embodiment, the methods of the invention are directed to subjectswho are “non-responsive” or “refractory” to a currently availabletherapy for PAH. In this context, the terms “non-responsive” and“refractory” refer to the subject's response to therapy as notclinically adequate to relieve one or more symptoms associated PAH. Theterms “subject” and “patient” are used interchangeably in this inventiondisclosure. The terms refer to an animal, preferably a mammal includinga non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and aprimate (e.g., a chimpanzee, a monkey such as a cynomolgous monkey and ahuman), and more preferably a human. In a preferred embodiment, thesubject is a human.

The terms “prevent”, “preventing” and “prevention” refer to theprevention of the recurrence, development, progression or onset of oneor more symptoms of PAH resulting from the administration of one or morecompounds identified in accordance the methods of the invention or theadministration of a combination of such a compound and a known therapyfor a disease or disorder.

Preferably, the administration of a composition according to the methodsof the invention in combination with one or more additional therapiesprovides a synergistic response in the subject having a disease ordisorder. In this context, the term “synergistic” refers to the efficacyof the combination being more effective than the additive effects ofeither single therapy alone. In one embodiment, the synergistic effectof combination rapamycin therapy according to the invention permits theuse of lower dosages and/or less frequent administration of at least onetherapy in the combination compared to its dose and/or frequency outsideof the combination. In another embodiment, the synergistic effect ismanifested in the avoidance or reduction of adverse or unwanted sideeffects associated with the use of either therapy in the combinationalone.

In the context of the pharmaceutical compositions of the invention, a“carrier” refers to, for example, a liquid or solid material such as asolvent, a diluent, stabilizer, adjuvant, excipient, auxiliary agent,propellant, or vehicle with which rapamycin is formulated for delivery.Examples of pharmaceutically acceptable carriers for use in thecompositions of the invention include, without limitation, dry powdercarriers such as lactose, mannose, amino acids, cyclodextrin,dipalmitylphosphatidylcholine, hydrocarbon and fluorocarbon propellants,compressed gases, sterile liquids, water, buffered saline, ethanol,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like), oils, detergents, suspending agents, carbohydrates(e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g.,ascorbic acid or glutathione), chelating agents, low molecular weightproteins, or suitable mixtures thereof. Preferably, in the context ofthe dry powder aerosol formulations of rapamycin, the carrier, ifpresent, is selected from the group consisting of a saccharide and asugar alcohol. In one embodiment, the carrier, if present, is lactose.

The term “pharmaceutically acceptable” indicates approval by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia such as theEuropean Pharmacopeia, for use in animals, and more particularly inhumans. One method for solubilizing poorly water soluble or waterinsoluble drugs is to form a salt of the drug or to prepare a prodrugthat is more soluble itself or that can be used to form a water solublesalt of the prodrug. Methods for forming salts and pharmaceuticallyacceptable salt forms are known in the art and include, withoutlimitation, salts of acidic or basic groups that may be present in thedrug or prodrug of interest. Compounds that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that can be used to prepare pharmaceuticallyacceptable acid addition salts of such basic compounds are those thatform non-toxic acid addition salts, i.e., salts containingpharmacologically acceptable anions, including but not limited tosulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, acid citrate,tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that areacidic in nature are capable of forming base salts with variouspharmacologically acceptable cations. Examples of such salts includealkali metal or alkaline earth metal salts and, particularly, calcium,magnesium, sodium lithium, zinc, potassium, and iron salts.

In one embodiment, the methods and compositions of the invention utilizea water soluble prodrug or derivative of rapamycin, preferablytemsirolimus or related compound. In one embodiment, the methods andcompositions of the invention utilize rapamycin (sirolimus).

Rapamycin

Rapamycin is a macrocyclic lactone produced by Streptomyceshygroscopicus. Its chemical (IUPAC) name is(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,-14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethox-y-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.

Its molecular formula is C₅₁H₇₉NO₁₃ and its molecular weight is 914.172g/mol. Its structure is shown below. Isomers of rapamycin are known,e.g., isomer B and isomer C, having structures as shown in U.S. Pat. No.7,384,953. Typically, rapamycin is a mixture of the B and C isomers. Insolution, rapamycin isomers B and C interconvert and an equilibrium isachieved. It is common practice in the literature to depict thestructure of rapamycin in the form of the B isomer, which is in the formshown below.

Rapamycin is a white to off-white powder and is considered insoluble inwater, having a very low solubility of only 2.6 μg/ml. It is freelysoluble in benzyl alcohol, chloroform, acetone, and acetonitrile. Thewater insolubility of rapamycin presents special technical problems toits formulation. In the context of its formulation as an oral dosageform, it has been prepared as an oral solution in the form of a soliddispersion (WO 97/03654) and a tablet containing nanosized (less than400 nm) particles (U.S. Pat. No. 5,989,591). But these procedures sufferfrom substantial variation in the dissolution of the active andtherefore its bioavailability. Another method of formulation utilizesthe crystalline powder. According to art-recognized methods, thetransformation of the crystalline form of a low solubility drug to itsamorphous form can significantly increase its solubility. While this isalso true for rapamycin, the amorphous form is extremely chemicallyunstable. Pharmaceutical dosage forms comprising amorphous rapamycin(sirolimus) are described in WO 06/039237 and WO 06/094507 (modifiedrelease formulation comprising sirolimus and glyceryl monostearate at aconcentration of 49.25%). An improved stable oral dosage form ofrapamycin is described in U.S. Pat. No. 8,053,444. The dosage formemploys a fatty acid ester and a polymer (e.g., polyvinylpyrrolidone(PVP), hydroxypropylcellulose (HPC) or hydroxypropylmethylcellulose(HPMC)) in the composition to increase the stability of sirolimuswithout adversely affecting its release rate. According to U.S. Pat. No.8,053,444, a fatty acid ester concentration exceeding 10% w/w suppressesthe release rate of sirolimus from the formulation and so should beavoided because it can lead to insufficient absorption from thegastrointestinal tract. The preferred concentration of fatty acid ester(glycerol ester) is 1% to 5% or 5% to 9%. In one embodiment, the aerosolrapamycin compositions of the present invention do not contain a fattyacid ester in combination with a polymer. In one embodiment, the aerosolrapamycin compositions of the invention contain a fatty acid ester at aconcentration exceeding 10% or exceeding 12% by weight of thecomposition.

Rapamycin and its derivatives (including analogs) and prodrugs suitablefor use in the compositions and methods of the invention includerapamycin (sirolimus) and prodrugs or derivatives thereof which areinhibitors of the mTOR cellular signaling pathway, and preferablyinhibitors of mTOR itself. In one embodiment, a rapamycin derivative orprodrug is an mTOR inhibitor selected from the group consisting ofeverolimus (Affinitor; RAD001), temsirolimus (CCI-779), ridaforolimus(previously known as deforolimus; AP23573), umirolimus (Biolimus A9),zotarolimus (ABT-578), novolimus, myolimus, AP23841, KU-0063794,INK-128, EX2044, EX3855, EX7518, AZD08055 and OSI027. Furtherderivatives are known to the skilled person and include, for example, anO-substituted derivative in which the hydroxyl group on the cyclohexylring of sirolimus is replaced by —OR₁, in which R₁ is optionally asubstituted alkyl, acylaminoalkyl or aminoalkyl.

In one embodiment, the compound for use in the aerosol formulations andmethods of the invention is a rapamycin derivative (analogue) selectedfrom the group consisting of analogues everolimus, temsirolimus,ridaforolimus, umirolimus, and zotarolimus. The chemical structures ofthe rapamycin analogues everolimus, temsirolimus, ridaforolimus,umirolimus, and zotarolimus are shown below.

In one embodiment, the compound for use in the aerosol formulations andmethods of the invention is an mTOR inhibitor selected from the groupconsisting of KU-0063794, AZD8055, INK128, and OSI-027. The chemicalstructures of the mTOR inhibitors KU-0063794, AZD8055, INK128, andOSI-027 are shown below.

Particularly preferred for use in the methods and compositions of theinvention are sirolimus, temsirolimus, and everolimus. In oneembodiment, the compound for use in the aerosol formulations and methodsof the invention is selected from the group consisting of sirolimus,temsirolimus, and everolimus. In one embodiment, the compound issirolimus or everolimus.

Compositions for Inhalation

The invention provides pharmaceutical compositions adapted foradministration by inhalation comprising rapamycin, or a prodrug orderivative thereof, in the form of an aqueous solution, a dry powder, ora mixture of one or more pharmaceutically acceptable propellants and acarrier. In one embodiment, the rapamycin is encapsulated in apharmaceutically acceptable compound, material, or matrix. In oneembodiment, the rapamycin is encapsulated in a liposomal formulation ora non-liposomal formulation.

The compositions of the invention are aerosolizable formulations ofrapamycin suitable for pulmonary drug delivery in a human subject byinhalation of the aerosol. The term “aerosol” is used in this context tomean a colloidal system in which the dispersed phase is composed ofeither solid or liquid particles and in which the dispersal medium is agas. In one embodiment, the gas is air and the formulation is a solutionformulation suitable for administration via a nebulizer or a dry powderformulation suitable for administration via dry powder inhaler device.Generally, respirable particles or droplets will have a mean diameter inthe range of 0.10 to 10 microns. The size of the particles or dropletsis selected to maximize targeted delivery either to the lungs themselves(i.e., where the lung is the target tissue) or systemically (where thelungs are utilized as an alternative route for systemic administration).The size will preferably be in the range of about 0.5 to 5 microns wherethe lung itself is the therapeutic target, or less than 3 microns forsystemic delivery via the lung. Size is measured according to methodsknown in the art and described, for example, in the U.S. Pharmacopeia atChapters 905 and 601. For example, it is measured as Mass MedianAerodynamic Diameter (MMAD). In one embodiment, the average or meandiameter of the particles comprising the compositions described hereinis measured as MMAD.

In one embodiment, the dispersed phase of the aerosol is composed ofliquid particles or droplets. In this context, the terms “liquidparticles” and “droplets” are used interchangeably. In this embodiment,the formulation of the invention is a solution formulation. In oneembodiment, the dispersed phase of the aerosol is composed of solidparticles. In this embodiment, the formulation of the invention is a drypowder formulation. Micronized particles of this size can be produced bymethods known in the art, for example by mechanical grinding (milling),precipitation from subcritical or supercritical solutions, spray-drying,freeze-drying, or lyophilization.

Generally, inhaled particles are subject to deposition by one of twomechanisms: impaction, which usually predominates for larger particles,and sedimentation, which is prevalent for smaller particles. Impactionoccurs when the momentum of an inhaled particle is large enough that theparticle does not follow the air stream and encounters a physiologicalsurface. In contrast, sedimentation occurs primarily in the deep lungwhen very small particles which have traveled with the inhaled airstream encounter physiological surfaces as a result of random diffusionwithin the air stream. The aerosol formulations of the invention arepreferably adapted to maximize their deposition either by impaction (inthe upper airways) or by sedimentation (in the alveoli), in order toachieve the desired therapeutic efficacy.

The amount of drug delivered to the patient from a delivery device, suchas a nebulizer, pMDI or DPI device, is referred to as the delivereddose. It can be estimated in vitro by determining the amount of drugemitted from the delivery device in a simulated inhalation maneuver.This is termed emitted dose (ED) as measured according to methods knownin the art, for examples those set out in the U.S. and EuropeanPharmacopeias, e.g., at Chapter 601 and Chapter 905 of the USP.Accordingly, “emitted dose” is considered equivalent to the delivereddose.

The amount of drug delivered from the delivery device to the lungs ofthe patient is termed the respirable dose. It can be estimated in vitroby determining the fine particle dose (FPD) as measured using cascadeimpactors, such as a Next Generation Impactor (NGI) according to methodsknown in the art, for examples those set out in the U.S. and EuropeanPharmacopeias, e.g., at Chapters 601 and 905 of the USP.

The amount of drug released in fine, inhalable particles from a deliverydevice is referred to as the fine particle fraction (FPF) of theformulation. FPF is the fraction of drug in the delivered dose that ispotentially respirable. Thus, FPF is the ratio of FPD to ED (emitted, ordelivered dose). These characteristics of the formulation are measuredaccording to methods known in the art, for examples those set out in theU.S. and European Pharmacopeias, e.g., at Chapter 601 of the USP andmonograph 2.9.18 of the Pharm Europa.

In one embodiment, the aerosolizable rapamycin formulations of thepresent invention have an FPF greater than 20% with a corresponding FPDranging from 10 micrograms to 2 milligrams, preferably less than 0.5milligrams, even after prolonged storage, e.g., after 1 to 12 months orafter 1 to 36 months of storage. In one embodiment the dose delivered tothe patient, the delivered dose (DD) or emitted dose (ED), ranges from25 micrograms to 2.5 milligrams, preferably less than 0.5 milligrams.

In certain embodiments the rapamycin is encapsulated in apharmaceutically acceptable compound, material, or matrix. In oneembodiment, the rapamycin is encapsulated in a liposomal formulation ornon-liposomal formulation.

Aqueous Solution Compositions

In one embodiment, the aerosolizable composition of the invention is anaqueous solution formulation of rapamycin adapted for pulmonary deliveryvia a nebulizer, including jet, vibrating mesh, and static mesh ororifice nebulizers. Thus, the solution formulation is adapted to enableaerosol droplet formation in the respirable range of from about 0.1 to10 micron diameter, as described above. In one embodiment, thecomposition is a nebulizable aqueous solution formulation consisting ofrapamycin (sirolimus) or a prodrug or derivative thereof, dissolved inwater, ethanol, and a low molecular weight polyol, and optionallyincluding a surface active agent. In one embodiment, the aqueoussolution formulation has a viscosity below 20 mPa-s, below 10 mPa-s, orbelow 5 mPa-s, and a surface tension of at least 45 dynes/cm, preferablygreater than 60 dynes/cm. Preferably, the formulation has a viscositybelow 5 mPa-s, and a surface tension above 45 dynes/cm. In oneembodiment, the composition has a viscosity below 20 mPa-s, a viscositybelow 10 mPa-s, or a viscosity below 5 mPa-s and a surface tension of atleast 45 dynes/cm, preferably greater than 60 dynes/cm.

In one embodiment, the aqueous solution formulation consists ofrapamycin, water, ethanol, and a low molecular weight polyol selectedfrom glycerol and propylene glycol. In one embodiment, the aqueoussolution formulation consists of rapamycin, water, and a low molecularweight polyol selected from glycerol and propylene glycol, with theethanol being optional. The formulation may also optionally contain anon-ionic surfactant, preferably PEG 100, or a polysorbate, preferablyPolysorbate 80 (“PS80”), a phospholipid, preferably a naturalphospholipid such as lecithin, and preferably hydrogenated soyalecithin, and an antioxidant or stabilizer, preferably disodium EDTA. Inone embodiment, the non-ionic surfactant is selected from the groupconsisting of polyethylene glycol (PEG) PEG 100, PEG 1000, andPolysorbate 80 (also referred to as Tween™ 80, sorbitan monooleate, orpolyoxyethylene sorbitan oleate), and mixtures thereof.

The amount of rapamycin in the aqueous solution is from about 0.001% to0.01% weight percent (% wt or % w/w) based on the total weight of thesolution. In one embodiment, rapamycin is present in solution at aconcentration of about 0.01 mg/ml to about 0.1 mg/ml. In one embodiment,the amount of rapamycin is from 0.001% to 0.01% w/w based upon totalweight of the solution.

In one embodiment, the concentration of rapamycin in solution is fromabout 0.01 to 0.1 mg/ml, the amount of the low molecular weight polyolis from 5 to 35% w/w, the amount of ethanol is present in the amount of5-20% w/w, and the amount of the non-ionic surfactant is from 1 to 200parts per million (ppm) w/w. Preferably, the amount of non-ionicsurfactant is less than 100 ppm (w/w). The amounts of the optionalantioxidant/stabilizer from zero to less than 0.01% w/w.

In one embodiment, the aqueous solution formulation of the inventiondoes not contain one or more additives or excipients selected from thegroup consisting of polyethylene glycol, lecithin, EDTA, a blockcopolymer, and a cyclodextrin.

The aqueous solution formulation is a single phase aqueous solution inwhich the rapamycin is completely dissolved. The main co-solvents in theformulation are ethanol and a low molecular weight polyol selected fromglycerol and propylene glycol. The rapamycin is not in suspension oremulsion, nor can the solution be described as a colloidal solution ordispersion. The aqueous solution formulation of the invention lackscolloidal structures such as micelles or liposomes. The amount ofphospholipid, if present, is too small to form liposomes or toprecipitate the rapamycin. And the combined amount of phospholipid andnon-ionic surfactant is too small to modify surface tension.Consequently, neither the phospholipid nor the non-ionic surfactant ispresent in amounts sufficient to act as a surfactant in the traditionalsense. In this context, the term surfactant refers to an agent that actsto lower the surface tension of the solution or the interfacial tensionbetween the liquid and any solid drug particles in solution such thatthe surfactant acts as a detergent, wetting agent, emulsifier, ordispersing agent. Instead, the non-ionic surfactant in the solutionformulation of the invention serves to block adsorption of the drug tothe polyethylene container in which the final product is packaged,thereby preventing loss of drug potency via adsorption to the container.

Accordingly, in one embodiment the aqueous solution formulation is asingle phase aqueous solution in which the rapamycin is completelydissolved, the solution lacks micelles or liposomes, and the solution isnot an emulsion, dispersion, or suspension.

In one embodiment, the solution formulation is sterile. In oneembodiment, the solution formulation is sterile filtered through a 0.2micron filter. In one embodiment, the solution formulation is notsterilized by heat, such as by autoclaving, or by radiation.

In one embodiment, the invention provides a package containing one ormore containers or vials (these terms are used interchangeably) filledwith the sterile aqueous solution formulation. Preferably, thecontainers are unit dose containers. In one embodiment, the containersare polymer vials, preferably polyethylene vials. In one embodiment, thecontainer or vial filled with the sterile aqueous solution formulationof the invention is produced by a process comprising the steps offorming the vial by blow molding and immediately thereafter filling thevial with the sterile-filtered formulation of the invention underaseptic conditions, followed by thermal sealing of the vial immediatelyafter it is filled.

In one embodiment, the aqueous aerosol formulation of the inventioncomprises or consists of the following

-   -   rapamycin (or a prodrug or derivative thereof) from about 0.001%        to 0.01% w/w,    -   propylene glycol from about 5% to 35% w/w,    -   ethanol from about 5% to 20% w/w,    -   Polysorbate 80 from about 1 to 200 ppm w/w,    -   lecithin from about 1 to 100 ppm w/w, and    -   water,        where the amount of water is sufficient to achieve a        concentration of the rapamycin between and 0.01 to 0.1        milligrams/milliliter. Optionally, a stability enhancer could be        added such as disodium EDTA at levels below 0.01% wt/wt.

For aqueous and other non-pressurized liquid systems, a variety ofnebulizers (including small volume nebulizers) are available toaerosolize the formulations. Compressor-driven nebulizers incorporatejet technology and use compressed air to generate the liquid aerosol.Such devices are commercially available from, for example, HealthdyneTechnologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.;Pari Respiratory, Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco,Inc., DeVilbiss Health Care, Inc.; and Hospitak, Inc. Ultrasonicnebulizers rely on mechanical energy in the form of vibration of apiezoelectric crystal to generate respirable liquid droplets and arecommercially available from, for example, Omron Healthcare, Inc. andDeVilbiss Health Care, Inc.

In one embodiment, the aqueous aerosol formulation of the invention isdelivered via a vibrating nebulizer available from Aerogen, Pari,Philips, or Omron. In one embodiment, the aqueous aerosol formulation ofthe invention is packaged in a container suitable for use with avibrating mesh nebulizer, for example, the Aeroneb® Go (Aerogen,distributed by Philips Respironics), I-Neb® (Philips) or E-Flow® (Pari),or similar nebulizer. In one embodiment the aqueous aerosol formulationof the invention is delivered via an orifice nebulizer such as theRespimat® from Boeringher-Ingelheim.

Thus, in one embodiment the invention provides a pharmaceuticalcomposition in the form of a nebulizable aqueous solution suitable foradministration by inhalation to a human subject, the aqueous solutionconsisting of rapamycin or a prodrug or derivative thereof, preferablyselected from sirolimus, everolimus, and temsirolimus, water, ethanol,and a low molecular weight polyol. In one embodiment, the low molecularweight polyol is glycerol or propylene glycol, or a mixture thereof. Inone embodiment, the composition further comprises a nonionic surfactantselected from the group consisting of PEG 100, PEG 1000, and polysorbate80, and mixtures thereof. In one embodiment, the amount of nonionicsurfactant in the formulation is from 1 to 200 ppm w/w, preferably lessthan 100 ppm w/w, based upon the weight of the formulation. In oneembodiment, the composition further comprises a phospholipid, anantioxidant or chemical stabilizer. In one embodiment, the amount ofantioxidant or chemical stabilizer in the formulation is less than 0.01%w/w based upon the weight of the formulation. In one embodiment, theantioxidant or chemical stabilizer is EDTA. In one embodiment, theamount of rapamycin in the formulation is from 0.001 to 0.01% w/w basedupon the weight of the formulation.

In one embodiment, the composition does not contain one or moreadditives or excipients selected from the group consisting ofpolyethylene glycol, lecithin, EDTA, a block copolymer, and acyclodextrin.

In one embodiment, the composition lacks colloidal structures selectedfrom micelles and liposomes.

In one embodiment, the composition is suitable for administration viaany one of a jet nebulizer, a vibrating mesh nebulizer, a static meshnebulizer, and an orifice nebulizer.

In one embodiment, the composition has a viscosity below 20 mPa-s,preferably below 10 mPa-s, most preferably below 5 mPa-s, and a surfacetension of at least 45 dynes/cm, preferably at least 50 dynes/cm.

The invention also provides a method of manufacturing a pharmaceuticalcomposition of the invention in the form of a nebulizable aqueoussolution, the method comprising sterile filtering the solution through afilter with pore size of 0.2 microns or less and collecting the sterilefiltrate in collection vessel under aseptic conditions. In oneembodiment, the method of manufacturing further comprises transferringthe sterile filtrate into a container closure under aseptic conditions.In one embodiment, the container closure is a unit-dose polyethylenevial. In one embodiment, the vial is produced by blowmolding immediatelybefore the sterile filtrate is transferred to the vial. In oneembodiment, the method further comprises the step of thermally sealingthe vial immediately after the sterile filtrate is transferred to thevial.

Dry Powder Compositions

In one embodiment, the aerosolizable composition of the invention is adry powder comprising micronized particles of rapamycin, or a prodrug orderivative thereof, as the therapeutic agent (also referred to as“drug”), the particles having diameters from 0.1 to 10 microns and amean diameter of between about 0.5 to 4.5 microns, about 1 to 4 microns,about 1 to 3.5 microns, about 1.5 to 3.5 microns, or about 2 to 3microns. The dry powder formulation is suitable for use in either a drypowder inhaler device (DPI) or a pressurized metered dose inhaler(pMDI). The amount of rapamycin in the dry powder is from about 0.5 to20% (w/w) based on total weight of the powder. In one embodiment, theamount of rapamycin is about 1% or 2% (w/w).

In one embodiment, micronized rapamycin is produced by wet polishing orjet milling as described below to generate diameters in the range ofabout 0.5 to 4.5 microns, about 1 to 4 microns, or about 2 to 3 microns,and the rapamycin particles are blended onto lactose carrier particlesin a drug/carrier ratio ranging from 0.5-2% w/w with a preferred ratioof 1%.

In one embodiment, the drug particles are lightly compacted into afrangible matrix which is contained within the delivery device (a drypowder inhaler). Upon actuation the delivery device abrades a portion ofthe drug particles from the matrix, and disperses them in theinspiratory breath delivering the drug particles to the respiratorytract. Alternatively the drug particles may be a free flowing powdercontained within a reservoir in the delivery device (a dry powderinhaler). The reservoir can be an integral chamber within the device, ora capsule, blister or similar preformed reservoir that is inserted intothe device prior to actuation. Upon actuation the device dispersed aportion of the drug particles from the reservoir and disperses them inthe inhalation breath delivering the drug particles to the respiratorytract.

In one embodiment, the dry powder composition consists of drug particlesand a carrier selected from the group consisting of arabinose, glucose,fructose, ribose, mannose, sucrose, trehalose, lactose, maltose,starches, dextran, mannitol, leucine, lysine, isoleucine,dipalmitylphosphatidylcholine, lecithin, polylactic acid, poly(lactic-co-glutamic) acid, and xylitol, or mixtures of any of theforegoing. In one embodiment, the carrier is lactose, particularly inthe form of the monohydrate. In one embodiment, the dry powdercomposition comprises a blend of two or more carriers.

In one embodiment the dry powder composition comprises drug and a blendof at least two different carriers. In one embodiment, the drug tocarrier ratio is in the range of from about 0.5 to 20% (w/w). In oneembodiment, the drug particles have diameters ranging from 0.1 to 10microns with a mean diameter of about 1 to 4, 1 to 3.5, or 1.5 to 3.5,or 2 to 3 microns. The carrier particles may have diameters ranging from2 to 200 microns.

In one embodiment, the composition is contained in a blister pack or areservoir of a DPI device. In one embodiment, the dry powder compositionis preloaded into a gelatin, starch, cellulosic, or polymeric capsule,or a foil/foil or foil/plastic blister suitable for use in a DPI device.Each capsule or blister may contain from 1 to 100 milligrams of the drypowder composition. The capsules or blisters may be inserted into a drypowder inhaler (DPI) device such as Aerolizer®, Plastiape® RS01 Model 7,and Plastiape® RS00 Model 8, XCaps®, FlowCaps®, Arcus®, Diskhaler® orMicrodose®. Upon actuating the DPI device, the capsules or blisters areruptured and the powder is dispersed in the inspiratory breath,delivering the drug to the respiratory tract.

In one embodiment, the dry powder composition is contained in a drypowder inhaler (DPI) device selected from Accuhaler®, Conix™,Rotahaler®, TwinCaps®, XCaps®, FlowCaps®, Turbuhaler®, NextHaler®,CycloHaler®, Revolizer™, Diskhaler®, Diskus®, Spinhaler, Handihaler®,Microdose Inhaler, GyroHaler®, OmniHaler®, Clickhaler®, Duohaler®(Vectura), and ARCUS® inhaler (Civitas Therapeutics). In one embodiment,the invention provides a DPI device containing a dry powder compositiondescribed herein. In one embodiment the device is selected from thegroup consisting of XCaps, FlowCaps, Handihaler, TwinCaps, Aerolizer®,Plastiape® RS01 Model 7, and Plastiape® RS00 Model 8. In one embodiment,the device containing the composition is selected from the groupconsisting of a GyroHaler®, an OmniHaler®, a Clickhaler®, a Duohaler®,and an ARCUS® inhaler.

The carrier particles are preferably of larger size (greater than 5microns) so as to avoid deposition of the carrier material in the deeplung. In one embodiment, the carrier particles have diameters rangingfrom 1 to 200 microns, from 30 to 100 microns, or less than 10 microns.In one embodiment the carrier particles are a blend of two carriers, onewith particles of about 30-100 microns and the other with particles lessthan 10 microns. The ratio of the two different carriers is in the rangeof from 3:97 to 97:3. In one embodiment, the dry powder compositionconsists of 0.5-20% (w/w) drug to carrier ratio, the drug particleshaving diameters from 0.1 to 10 microns with a mean diameter less than3.5 microns. In one embodiment, the carrier material is a crystallinecarrier material. Preferably, the crystalline carrier material is onewhich is at least 90%, preferably greater than 95% crystalline and inwhich no or substantially no water is absorbed by the carrier underconditions of 80% or lower relative humidity at room temperature.Examples of such crystalline carriers are lactose monohydrate andglucose monohydrate. The amount of carrier is from 1 to 99.0% or more ofthe formulation by dry weight of the powder, preferably 5 to 99%, 10 to99%, 20 to 99%, 30 to 99%, 40 to 99%, or 50 to 99%.

In one embodiment, the dry powder composition is contained within areservoir in the delivery device (a dry powder inhaler). The reservoircan be an integral chamber within the device, or a capsule, blister orsimilar preformed reservoir that is inserted into the device prior toactuation. Upon actuation the device dispersed a portion of the drugparticles from the reservoir and disperses them in the inspiratorybreath delivering the drug particles to the respiratory tract.

In one embodiment, drug is present as a fine powder with apharmaceutically acceptable carrier. In the context, the term “fine”refers to a particle size in the inhalable range, as discussed above.Preferably, the drug is micronized such that the particles have a meandiameter in the range of 10 microns or less. In one embodiment, the meandiameter (MMAD or Dv50) of the particles of rapamycin (or a prodrug orderivative thereof) in dry powder composition described herein is from0.5 to 10 microns, from 0.5 to 6 microns, from 1 to 5 microns, from 1 to4 microns, from 1 to 3 microns, or from 2 to 3 microns. The MMAD or Dv50value is the particle size below which 50% of the volume of thepopulation occurs.

In one embodiment, the dry powder formulation of rapamycin furthercomprises one or more additives selected from the additives describedbelow. In one embodiment, the one or more additives comprises orconsists of magnesium stearate. In one aspect of this embodiment, themagnesium stearate is present in amounts of 0.001 to 10% by dry weightof the powder, preferably in amounts of from 0.01 to 5% or 0.01 to 2%.In another embodiment, the additive comprises or consists of aphospholipid, such as lecithin (which is a mixture ofphosphatidylcholines) in an amount of 0.1% to 1% by dry weight of thepowder, preferably 0.2% to 0.6%. In one aspect of this embodiment, theadditive is coated onto the carrier material prior to or simultaneouslywith a step of blending the carrier with the particles of rapamycin.This can be accomplished, for example, by utilizing a high energy mixingstep to coat the carrier with the additive, or a long duration of lowenergy mixing, or a combination of low and high energy mixing to achievethe desired level of coated carrier material. Low energy devices formixing dry powders to form blends are known in the art and include, forexample, V-blenders, double cone blenders, slant cone blenders, cubeblenders, bin blenders, horizontal or vertical drum blenders, staticcontinuous blenders, and dynamic continuous blenders. Other, higherenergy devices include high shear mixers known to those skilled in theart.

In certain embodiments, the dry powder is contained in a capsule. In oneembodiment the capsule is a gelatin capsule, a plastic capsule, or acellulosic capsule, or is in the form of a foil/foil or foil/plasticblisters. In each instance, the capsule or blister is suitable for usein a DPI device, preferably in dosage units together with the carrier inamounts to bring the total weight of powder in each capsule to from 1 mgto 100 mg. Alternatively, the dry powder may be contained in a reservoirof a multi-dose DPI device.

The particle size of the rapamycin can be reduced to the desiredmicroparticulate level by conventional methods, for example by grindingin an air-jet mill, ball mill or vibrator mill, by wet polishing,microprecipitation, spray drying, lyophilization or recrystallizationfrom subcritical or supercritical solutions. Jet milling or grinding inthis context refers to micronization of dry drug particles by mechanicalmeans. Micronization techniques do not require making a solution,slurry, or suspension of the drug. Instead, the drug particles aremechanically reduced in size. Due to the relatively high energy that isemployed by micronization, in certain embodiments it is desirable toinclude a carrier material in a co-micronized mixture with therapamycin. In this context, the carrier material absorbs some of theenergy of micronization which otherwise could adversely affect thestructure of the rapamycin. In one embodiment, rapamycin particles in asize range of from 1 to 4 or from 2 to 3 microns are produced by a jetmilling method.

Wet polishing as described in US2013/0203717 involves using high shearto reduce the particle size of the drug particles in a suspension orslurry. Wet polishing can include just the drug particles or additionalparticulates termed milling media. In one embodiment, the particle sizeof the rapamycin can be reduced to the desired level using a wetpolishing process, which comprises wet milling, specifically bycavitation at elevated pressure, where rapamycin is suspended in wateror other solvent where it is insoluble, and then is followed by spraydrying of the suspension to obtain rapamycin as a dry powder. In oneembodiment, rapamycin particles in a size range of from 1 to 4 or from 2to 3 microns are produced by a wet polishing method that comprisespreparing a suspension of rapamycin, subjecting the suspension tomicrofluidization, and spray-drying the resulting particles to form adry powder. The rapamycin may be suspended in an anti-solvent selectedfrom the group consisting of propyl or butyl alcohol, water, and ethylacetate. In one embodiment, the suspension is an aqueous suspension.

Spray drying generally involves making a solution, slurry, or suspensionof the drug, atomizing the solution, slurry, or suspension, to formparticles and then evaporating the solution, slurry, or suspension mediato form the particles. The solution, slurry or suspension, can be formedunder subcritical or supercritical conditions. The evaporation step canbe accomplished by elevating the temperature of the atmosphere intowhich the atomization occurs, or by decreasing the pressure, or acombination of both. In one embodiment, the powder formulationcomprising rapamycin is made by spray drying an aqueous dispersion ofrapamycin to form a dry powder consisting of aggregated particles ofrapamycin having a size suitable for pulmonary delivery, as describedabove. The aggregate particle size can be adjusted (increased ordecreased) to target either the deep lung or upper respiratory sites,such as the upper bronchial region or nasal mucosa. This can beaccomplished, for example, by increasing the concentration of rapamycinin the spray-dried dispersion or by increasing the droplet sizegenerated by the spray dryer.

Alternatively, the dry powder can be made by freeze-drying(lyophilization) the aqueous drug solution, dispersion, or emulsion, orby a combination of spray-drying and freeze-drying.

In one embodiment, the aqueous dispersion of rapamycin and the one ormore optional additives further comprises a dissolved diluent such aslactose or mannitol such that when the dispersion is freeze-dried,respirable diluent particles, each containing at least one embedded drugparticle and additive particle, if present, are formed.

In one embodiment, the dry powder formulation is made by freeze-dryingan aqueous dispersion of rapamycin, and one or more optional additives.In one embodiment, the powders contain aggregates of rapamycin and anadditive, if present, wherein the aggregates are within a respirablesize range as described above.

In one embodiment, the dry powder comprises rapamycin loaded liposomes.Drug-loaded liposomes can be produced by methods known in the art, forexample using the technique described for tacrolimus in M. Chougale, etal. Int. J. Nanomedicine 2:625-688 (2007). Briefly, rapamycin,hydrogenated phosphatidylcholine (HSPC), and cholesterol are dissolvedin a mixture of methanol and chloroform and then subjected to dry thinfilm formation, e.g., in Rotaevaporator. The liposomes are hydrated andthe liposomal dispersion is passed through a high-pressure homogenizerfor size reduction. The resultant pellets are characterized for vesiclesize and percent drug entrapment and pellets equivalent to the desiredamount of rapamycin are then dispersed in a suitable medium andsubjected to spray-drying to obtain particles of the desired size forinhalation. The spray dried powder can be filled into capsules,canisters, or blister packs for administration.

In one embodiment the dry powder particles can be produced byprecipitation from a supercritical or subcritical solution.

The dry powder compositions may be contained in a suitable dry powderinhaler device, or in a capsule or blister for use in such a device.Examples of such devices are provided above and include Accuhaler®,Aerolizer®, the Plastiape® RS01 Model 7, the Plastiape® RS00 Model 8,Conix™, Rotahaler®, TwinCaps®, XCaps®, FlowCaps®, Turbuhaler®,NextHaler®, CycloHaler®, Revolizer™, Diskhaler®, Diskus®, Spinhaler,Handihaler®, Microdose Inhaler, GyroHaler®, OmniHaler®, Clickhaler®, orDuohaler® (Vectura), or a breath-actuated ARCUS® inhaler (CivitasTherapeutics). In one embodiment, the invention provides a DPI devicecontaining a dry powder composition described herein. In one embodimentthe device is selected from the group consisting of XCaps, FlowCaps,Handihaler, TwinCaps, Aerolizer®, the Plastiape® RS01 Model 7, and thePlastiape® RS00 Model 8.

Propellant-Based Formulations

In another embodiment of the invention, the rapamycin is formulated in apropellant-based formulation which may also be referred to genericallyherein as “a pMDI formulation”. A pMDI formulation is suitable fordelivery by a device such as a pressurized metered dose inhaler (pMDI).In one embodiment, the composition comprises rapamycin, a propellant,and a vegetable oil or pharmaceutically acceptable derivative of avegetable oil. The propellant is preferably selected from1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafluoropropane(HFA227), or mixtures thereof. In one embodiment, the vegetable oil isselected from olive oil, safflower oil, and soybean oil. The rapamycinmay be in solution or in suspension in the propellant. In this context,“in suspension” refers to where the rapamycin is present in particulateform dispersed in the propellant. In one embodiment, the rapamycin ismicronized and is present in suspension in the propellent. In oneembodiment, the formulation further comprises a wetting agent orco-solvent such as ethanol. In one embodiment, the formulation furthercomprises a polyhydroxy alcohol such as propylene glycol.

Suitable propellants are known in the art and include, for example,halogen-substituted hydrocarbons, for example fluorine-substitutedmethanes, ethanes, propanes, butanes, cyclopropanes or cyclobutanes,particularly 1,1,1,2-tetrafluoroethane (HFA134a) and1,1,1,2,3,3,3-heptafluoropropane (HFA227), or mixtures thereof.

In one embodiment, the formulation comprises micronized rapamycin,ethanol, a suitable propellant such as HFA 134a, HFA 227, or a mixtureof suitable propellants, and optionally one or more surfactants. In oneembodiment, the formulation further comprises a lubricant.

In one embodiment, the formulation comprises rapamycin, a propellant,and a vegetable oil. In one aspect, the formulation does not comprise anadditive or surfactant. For example, the formulation does not compriseethanol, a polyhydroxy alcohol (e.g., propylene glycol), or a surfactant(e.g., sorbitan trioleate, sorbitan monooleate, or oleic acid).

In one embodiment, the propellant-based formulation comprises compressedair, carbon dioxide, nitrogen or a liquefied propellant selected fromthe group consisting of n-propane, n-butane, isobutane or mixturesthereof, or 1,1,1,2-tetrafluoroethane (HFA134a) and1,1,1,2,3,3,3-heptafluoropropane (HFA227), or mixtures thereof, with orwithout a polar co-solvent such as an alcohol. The composition can be asolution or a suspension. For suspensions the drug particles havediameters from 0.1 to 10 microns with a mean diameter less than 3.5microns.

The propellant-based formulation is prepared by methods known in theart, for example by wet milling the coarse rapamycin, and optionaladditive, in liquid propellant, either at ambient pressure or under highpressure conditions. In certain embodiments, the additive is asurfactant which serves to prevent aggregation (caking orcrystallization), to facilitate uniform dosing, and (or alternatively)to provide a favorable fine particle fraction (FPF). In one aspect, thesurfactant is selected from sorbitan trioleate, sorbitan monooleate, oroleic acid. Alternatively, dry powders containing drug particles areprepared by spray-drying or freeze-drying aqueous dispersions of thedrug particles as discussed above and the resultant powders dispersedinto suitable propellants for use in conventional pressurized metereddose inhalers (pMDIs). In one embodiment, the inhalation device is aRespimat™.

In one embodiment, the propellant-based aerosol rapamycin formulationsof the invention are stable against particle size growth or change inthe crystal morphology of the rapamycin over prolonged periods of time.

Process for Manufacturing Sterile Unit Dose Forms

In one embodiment, the compositions of the invention are sterilecompositions. In one embodiment, the sterile compositions are sterileunit dose forms. In one embodiment, the sterile unit dosage form is acapsule suitable for use in a nebulizer device.

In one embodiment, the finished composition is sterilized in itscontainer-closure by heat, e.g., autoclaving, or by radiation. In oneembodiment, the component parts of the composition are first sterilizedby a suitable process including sterile filtration for liquid componentsand radiation or autoclaving for solids or liquids, the process furthercomprising maintaining the sterility of the sterile components bypackaging in hermetic containers, combining the components in a mixingvessel in the appropriate proportions, and filling the resulting productinto a container closure, all performed in an aseptic suite. Thisprocess has the disadvantage of being expensive and requiring difficultaseptic handling techniques. Accordingly, it is used primarily toprocess particulate suspensions or colloidal dispersions, liposomalformulations, or emulsions, which cannot be passed through a submicronfilter for sterilization. Finally, in one embodiment, the finishedcomposition is sterile filtered through a submicron filter, preferably a0.2 micron filter. In one embodiment, the compositions of the inventionare single-phase aqueous solutions sterilized via a filtrationsterilization process. In contrast, emulsions and liposomal formulationsare typically not sufficiently stable under the high shear conditions ofa filtration sterilization process and so are not preferred for thisprocess.

In one embodiment, the compositions of the invention are single-phaseaqueous solutions which are filled into a container-closure, e.g., avial, formed of a polymer, preferably polyethylene, or alternatively aglass vial. Autoclaving and radiation are not suitable where the vial isa polymer vial because of the high likelihood of creating chemicalinstability in the drug and/or formulation excipients, as well as in thecontainer, and due to the generation of undesirable impurities. In oneembodiment, the compositions of the invention are sterilized by aprocess that does not include heat (autoclaving) or radiation, andinstead includes a filtration sterilization process. Preferably, inaccordance with this embodiment, the single-phase aqueous solutions ofrapamycin are sterilized by filtration through a filter having a poresize less than or equal to 0.2 microns. In one embodiment, the sterilefiltrate is collected in a collection vessel located in an asepticsuite. In one embodiment, the sterile filtrate is transferred from thecollection vessel into a container closure in an aseptic suite.Preferably the container closure is a polymer vial, preferably a unitdose vial, and most preferably a polyethylene unit dose vial. In oneembodiment, the polymer vial is formed by blowmolding immediately beforeit is filled and then thermally sealed immediately after filling. Thistechnique may be also referred to as “form-fill-seal” or a “blow-fill”.This technique is particularly advantageous in the context of thecompositions of the invention which are single-phase aqueous solutionsof rapamycin because this process does not require heat or radiation,both of which may degrade either the drug itself, the formulationexcipients, or the container closure.

Pulmonary Administration and Dosing

The present invention provides compositions and methods for thetreatment and prophylaxis of PAH in a human subject by administeringrapamycin to the respiratory tract, preferably to the lungs, byinhalation. Pulmonary delivery is preferably accomplished by inhalationof the aerosol through the mouth and throat into the lungs, but may alsobe accomplished by inhalation of the aerosol through the nose. Thus, inone embodiment the composition is delivered intranasally. In anotherembodiment, the aerosol is delivered perorally.

The compositions and methods of the invention advantageously provide forthe targeted delivery of a therapeutically effective amount of rapamycinto the lungs while simultaneously reducing to very low or undetectablelevels the amount of rapamycin in the blood and available systemically.In one embodiment, the amount of rapamycin in a single dose of a drypowder composition described herein is from about 5 to 500 micrograms orfrom about 100 to 300 micrograms, or from about 50 to 250 micrograms.The targeted delivery of low dose rapamycin directly to the lungs whileminimizing systemic exposure provides for an improved therapeutic indexcompared to oral dosage forms.

In one embodiment, administration of rapamycin by inhalation accordingto the methods of the invention increases the therapeutic index ofrapamycin. In this context, as applied to human subjects, thetherapeutic index is a ratio that compares the dose that produces atherapeutic effect (ED₅₀) to the dose that produces a toxicity (TD₅₀) in50% of the population. The ratio is represented as TD₅₀/ED₅₀. In oneembodiment, administration of rapamycin by inhalation according to themethods of the invention reduces one or more toxicities associated withorally administered rapamycin, thereby increasing the therapeutic indexof rapamycin.

The invention includes aerosolizable formulations in the form ofsolutions and powders. Accordingly, the rapamycin may be administeredaccording to the methods of the invention in the form of an aqueousaerosol, a dry powder aerosol, or a propellant-based aerosol.

In one embodiment, the administered dose of rapamycin produces a bloodtrough level in the subject of from of from 0.01 to 0.15 ng/ml, from0.075 to 0.350 ng/ml, from 0.150 to 0.750 ng/ml, from 0.750 to 1.5 ng/mlor from 1.5 to 5 ng/ml. In one embodiment, the administered dose ofrapamycin produces a blood trough level in the subject of less than 5ng/ml, less than 2 ng/ml, less than 1 ng/ml, or less than 0.5 ng/ml.

In one embodiment, the administered dose of rapamycin is sufficient toproduce a concentration of rapamycin in lung tissue in the range of from1 ng/g to 1 ug/g, preferably from about 5 ng/g to 100 ng/g, from about 5ng/g to about 20 ng/g, or from about 5 ng/g to about 30 ng/g.

In one embodiment, the administered dose of rapamycin is from 5 to 100micrograms, from 20 to 100 micrograms, from 20 to 250 micrograms, from50 to 500 micrograms (0.05 to 0.5 milligrams), from 250 to 1000micrograms (0.25 to 1 milligrams) or from 500 to 2000 micrograms (0.5 to2 milligrams). In one embodiment, the amount of rapamycin administeredis less than 500 micrograms, less than 100 micrograms, less than 50micrograms, less than 20 micrograms, or less than 10 micrograms.Preferably, the amount of rapamycin administered is less than 0.5milligrams or less than 0.25 milligrams.

In one embodiment, the rapamycin is administered once daily.

In one embodiment, the total daily dose of rapamycin is in the range offrom 5 to 100 micrograms, from 20 to 250 micrograms, from 50 to 500micrograms (0.05 to 0.5 milligrams), from 250 to 1000 micrograms (0.5 to1 milligrams) or from 500 to 2000 micrograms (0.5 to 2 milligrams). Inone embodiment, the total daily dose of rapamycin is less than 500milligram, less than 100 micrograms, less than 50 micrograms, less than20 micrograms, or less than 10 microgram. In one embodiment, the totaldaily dose of rapamycin administered to the subject is less than 0.5milligrams or less than 0.25 milligrams per day.

In one embodiment, a composition of the invention is administered onceper day to the subject. In one embodiment, a composition of theinvention is administered twice or three times a day. Preferably, thecomposition is administered once or twice daily, or less than oncedaily.

In one embodiment, the methods of the invention comprise administeringrapamycin via a pulmonary route in combination with one or moreadditional therapeutic agents selected from the group consisting of aprostanoid, a phosphodiesterase inhibitor, or an endothelin antagonist.In one embodiment, the prostanoid is selected from the group consistingof a prostaglandin, a thromboxane, and a prostacyclin. The one or moreadditional agents may be administered by the same or a different routeof administration as the rapamycin. For example, the agent may beadministered by inhalation, intranasally, orally or intravenously.

In one embodiment, the methods of the invention comprise administeringrapamycin via a pulmonary route in combination with one or moreadditional therapies. In one embodiment, the one or more additionaltherapies is selected from oxygen therapy, vascodilation therapy,hormonal therapy, cardiovascular therapy, anti-proliferative therapy,autoimmune therapy, anti-inflammatory therapy, and anti-estrogentherapy.

In certain embodiments, the methods include pulmonary administration ofa composition of the invention as the primary therapy. In otherembodiments, the administration of a composition of the invention is anadjuvant therapy. In either case, the methods of the inventioncontemplate the administration of a composition of the invention incombination with one or more additional therapies for the treatment of adisease or disorder. The terms “therapy” and “therapies” refer to anymethod, protocol and/or agent that can be used in the prevention,treatment, management or amelioration of a disease or disorder, or oneor more symptoms thereof. In certain embodiments, a therapy is selectedfrom oxygen therapy, vascodilation therapy, hormonal therapy,cardiovascular therapy, anti-proliferative therapy, autoimmune therapy,anti-inflammatory therapy, and anti-estrogen therapy.

Preferably, the administration of a pharmaceutical compositioncomprising rapamycin or a prodrug or derivative thereof according to themethods of the invention in combination with one or more additionaltherapies provides a synergistic response in the subject having PAH. Inthis context, the term “synergistic” refers to the efficacy of thecombination being more effective than the additive effects of eithersingle therapy alone. In one embodiment, the synergistic effect ofcombination rapamycin therapy according to the invention permits the useof lower dosages and/or less frequent administration of at least onetherapy in the combination compared to its dose and/or frequency outsideof the combination. In another embodiment, the synergistic effect ismanifested in the avoidance or reduction of adverse or unwanted sideeffects associated with the use of either therapy in the combinationalone.

Nebulizer Delivery

In one embodiment, the rapamycin is formulated as an aqueous solutionsuitable for nebulization and delivered via a nebulizer. For aqueous andother non-pressurized liquid systems, a variety of nebulizers (includingsmall volume nebulizers) are available to aerosolize the formulations.Compressor-driven nebulizers incorporate jet technology and usecompressed air to generate the liquid aerosol. Such devices arecommercially available from, for example, Healthdyne Technologies, Inc.;Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari Respiratory,Inc.; Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc., DeVilbiss HealthCare, Inc.; and Hospitak, Inc. Ultrasonic nebulizers rely on mechanicalenergy in the form of vibration of a piezoelectric crystal to generaterespirable liquid droplets and are commercially available from, forexample, Omron Healthcare, Inc. and DeVilbiss Health Care, Inc. Thenebulizer may be, for example, a conventional pneumatic nebulizer suchas an airjet nebulizer, or an ultrasonic nebulizer, which may contain,for example, from 1 to 50 ml, commonly 1 to 10 ml, of the solutionformulation.

In one embodiment, the aqueous solution formulation of the invention isadapted for administration with a nebulizer comprising a vibrating orfixed mesh. For example, devices such as an AERx® (Aradigm), RESPIMAT®(Boehringer Ingelheim), I-Neb® (Philips), or MicroAire® (Omron) in whichdrug solution is pushed with a piston or pneumatic pressure, or with apiezoelectric crystal through an orifice or mesh. Alternatively, thesolution can be pumped through a vibrating mesh nebulizer such as theE-Flow® (Pari) or Aeroneb® Go (Aerogen). These devices allow muchsmaller nebulized volumes, e.g., 10 to 100 ul, and higher deliveryefficiencies than conventional nebulizers.

Dry Powder Delivery

In one embodiment, the dry powder compositions of the invention aredelivered by a non-propellant based dry powder inhaler (DPI) device. Inone embodiment, the powder is contained in capsules of gelatin orplastic, or in blisters, suitable for use in a DPI device. In oneembodiment, the powder is supplied in unit dosage form and in dosageunits of from 5 mg to 100 mg of powder per capsule. In anotherembodiment, the dry powder is contained in a reservoir of a multi-dosedry powder inhalation device. In one embodiment, the inhaler devicecomprises an aerosol vial provided with a valve adapted to deliver ametered dose, such as 10 to 100 μl, e.g. 25 to 50 μl, of thecomposition, i.e. a device known as a metered dose inhaler.

In one embodiment, the DPI device is a blister based device such as theGyroHaler® or the OmniHaler® (both from Vectura), a reservoir baseddevice such as the Clickhaler® or Duohaler® (Vectura), and the ARCUS®inhaler (Civitas Therapeutics). In one embodiment, the DPI device isselected from Pulmatrix™, and Hovione Twincaps and XCaps™. In oneembodiment the device is selected from the group consisting of XCaps,Plastiape® RS01 Model 7, and Plastiape® RS00 Model 8.

In one embodiment, the DPI device is selected from the group consistingof Accuhaler®, Aerolizer®, the Plastiape® RS01 Model 7, the Plastiape®RS00 Model 8, Conix™, Rotahaler®, TwinCaps®, XCaps®, FlowCaps®,Turbuhaler®, NextHaler®, CycloHaler®, Revolizer™, Diskhaler®, Diskus®,Spinhaler, Handihaler®, Microdose Inhaler, GyroHaler®, OmniHaler®,Clickhaler®, or Duohaler® (Vectura), or a breath-actuated ARCUS® inhaler(Civitas Therapeutics).

In one embodiment, the DPI device is selected from the group consistingof Arcus™, Aspirair™, Axahaler™, Breezhaler™, Clickhaler™, Conix Dry™,Cricket™, Dreamboat™, Genuair™, Gemini™, Inspiromatic™, iSPERSE™,MicroDose™, Next DPI™, Prohaler™, Pulmojet™, Pulvinal™, Solis™, Taifun™,Taper Dry™, Trivai™, Novolizer™, Podhaler™, Skyehaler™, Spiromax™,Twincaps/Flowcaps™, and Turbuhaler™. In one embodiment, the DPI deviceis adapted to deliver the dry powder from a capsule or blistercontaining a dosage unit of the dry powder or a multi-dose dry powderinhalation device adapted to deliver, for example, 5-25 mg of dry powderper actuation.

pMDI Delivery

In another embodiment, the rapamycin is delivered in the form ofaerosolized particles from a pressurized container or dispenser thatcontains a suitable propellant as described above in connection withpropellant-based formulations. In one embodiment, the inhaler is apropellant driven inhaler, such as a pMDI device, which releases ametered dose of rapamycin upon each actuation. A typical pMDI devicecomprises a canister containing drug, a drug metering valve, and amouthpiece. In one aspect of this embodiment, the rapamycin isformulated as a suspension in the propellant. In the context of thisembodiment, the rapamycin is made into a fine powder which is suspendedin the liquefied propellant or propellant blend. The suspension is thenstored in a sealed canister under sufficient pressure to maintain thepropellant in liquid form. In another embodiment, the rapamycin isformulated as a solution. In the context of this embodiment, therapamycin is solubilized in the liquefied propellant or propellantblend. In one embodiment, the formulation further comprises a stabilizerin an amount suitable to stabilize the formulation against settling,creaming or flocculation for a time sufficient to allow reproducibledosing of the rapamycin after agitation of the formulation. Thestabilizer may be present in excess in an amount of about 10 part byweight to about 5000 parts by weight based on one million parts by totalweight of the aerosol formulation. In one embodiment, the fluid carrieris 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane or amixture thereof. In another embodiment, the fluid carrier is ahydrocarbon (e.g., n-butane, propane, isopentane, or a mixture thereof).The composition may further comprise a co-solvent (e.g., ethanol orother suitable co-solvent).

In one embodiment of the methods of the invention, the aerosolformulation comprising rapamycin further comprises an additional drug.In one aspect of this embodiment, the additional drug is selected fromthe group consisting of corticosteroids, estrogen receptor antagonists,anticholinergics, beta-agonists, non-steroidal anti-inflammatory drugs,macrolide antibiotics, bronchodilators, leukotriene receptor inhibitors,muscarinic antagonists, cromolyn sulfate, and combinations thereof.

Additives

The aerosol compositions of the invention may contain one or moreadditives in addition to any carrier or diluent (such as lactose ormannitol) that is present in the formulation. In one embodiment, the oneor more additives comprises or consists of one or more surfactants.Surfactants typically have one or more long aliphatic chains such asfatty acids which enables them to insert directly into the lipidstructures of cells to enhance drug penetration and absorption. Anempirical parameter commonly used to characterize the relativehydrophilicity and hydrophobicity of surfactants is thehydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLBvalues are more hydrophobic, and have greater solubility in oils, whilesurfactants with higher HLB values are more hydrophilic, and havegreater solubility in aqueous solutions. Thus, hydrophilic surfactantsare generally considered to be those compounds having an HLB valuegreater than about 10, and hydrophobic surfactants are generally thosehaving an HLB value less than about 10. However, these HLB values aremerely a guide since for many surfactants, the HLB values can differ byas much as about 8 HLB units, depending upon the empirical method chosento determine the HLB value.

Among the surfactants for use in the aerosol compositions of theinvention are polyethylene glycol (PEG)-fatty acids and PEG-fatty acidmono and diesters, PEG glycerol esters, alcohol-oil transesterificationproducts, polyglyceryl fatty acids, propylene glycol fatty acid esters,sterol and sterol derivatives, polyethylene glycol sorbitan fatty acidesters, polyethylene glycol alkyl ethers, sugar and its derivatives,polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene(POE-POP) block copolymers, sorbitan fatty acid esters, ionicsurfactants, fat-soluble vitamins and their salts, water-solublevitamins and their amphiphilic derivatives, amino acids and their salts,and organic acids and their esters and anhydrides. Each of these isdescribed in more detail below.

PEG Fatty Acid Esters

Although polyethylene glycol (PEG) itself does not function as asurfactant, a variety of PEG-fatty acid esters have useful surfactantproperties. Among the PEG-fatty acid monoesters, esters of lauric acid,oleic acid, and stearic acid are most useful in embodiments of thepresent invention. Preferred hydrophilic surfactants include PEG-8laurate, PEG-8 oleate, PEG-8 stearate, PEG-9 oleate, PEG-10 laurate,PEG-10 oleate, PEG-12 laurate, PEG-12 oleate, PEG-15 oleate, PEG-20laurate and PEG-20 oleate. The HLB values are in the range of 4-20.

Polyethylene glycol fatty acid diesters are also suitable for use assurfactants in the compositions of embodiments of the present invention.Most preferred hydrophilic surfactants include PEG-20 dilaurate, PEG-20dioleate, PEG-20 distearate, PEG-32 dilaurate and PEG-32 dioleate. TheHLB values are in the range of 5-15.

In general, mixtures of surfactants are also useful in embodiments ofthe present invention, including mixtures of two or more commercialsurfactants as well as mixtures of surfactants with another additive oradditives. Several PEG-fatty acid esters are marketed commercially asmixtures or mono- and diesters.

Polyethylene Glycol Glycerol Fatty Acid Esters

Preferred hydrophilic surfactants are PEG-20 glyceryl laurate, PEG-30glyceryl laurate, PEG-40 glyceryl laurate, PEG-20 glyceryl oleate, andPEG-30 glyceryl oleate.

Alcohol-Oil Transesterification Products

A large number of surfactants of different degrees of hydrophobicity orhydrophilicity can be prepared by reaction of alcohols or polyalcoholwith a variety of natural and/or hydrogenated oils. Most commonly, theoils used are castor oil or hydrogenated castor oil, or an ediblevegetable oil such as corn oil, olive oil, peanut oil, palm kernel oil,apricot kernel oil, or almond oil. Preferred alcohols include glycerol,propylene glycol, ethylene glycol, polyethylene glycol, sorbitol, andpentaerythritol. Among these alcohol-oil transesterified surfactants,preferred hydrophilic surfactants are PEG-35 castor oil (Incrocas-35),PEG-40 hydrogenated castor oil (Cremophor RH 40), PEG-25 trioleate(TAGAT® TO), PEG-60 corn glycerides (Crovol M70), PEG-60 almond oil(Crovol A70), PEG-40 palm kernel oil (Crovol PK70), PEG-50 castor oil(Emalex C-50), PEG-50 hydrogenated castor oil (Emalex HC-50), PEG-8caprylic/capric glycerides (Labrasol), and PEG-6 caprylic/capricglycerides (Softigen 767). Preferred hydrophobic surfactants in thisclass include PEG-5 hydrogenated castor oil, PEG-7 hydrogenated castoroil, PEG-9 hydrogenated castor oil, PEG-6 corn oil (Labrafil® M 2125CS), PEG-6 almond oil (Labrafil® M 1966 CS), PEG-6 apricot kernel oil(Labrafil® M 1944 CS), PEG-6 olive oil (Labrafil® M 1980 CS), PEG-6peanut oil (Labrafil® M 1969 CS), PEG-6 hydrogenated palm kernel oil(Labrafil® M 2130 BS), PEG-6 palm kernel oil (Labrafil® M 2130 CS),PEG-6 triolein (Labrafil®b M 2735 CS), PEG-8 corn oil (Labrafil® WL 2609BS), PEG-20 corn glycerides (Crovol M40), and PEG-20 almond glycerides(Crovol A40).

Polyglyceryl Fatty Acids

Polyglycerol esters of fatty acids are also suitable surfactants for usein embodiments of the present invention. Among the polyglyceryl fattyacid esters, preferred hydrophobic surfactants include polyglyceryloleate (Plurol Oleique), polyglyceryl-2 dioleate (Nikkol DGDO),polyglyceryl-10 trioleate, polyglyceryl stearate, polyglyceryl laurate,polyglyceryl myristate, polyglyceryl palmitate, and polyglyceryllinoleate. Preferred hydrophilic surfactants include polyglyceryl-10laurate (Nikkol Decaglyn 1-L), polyglyceryl-10 oleate (Nikkol Decaglyn1-0), and polyglyceryl-10 mono, dioleate (Caprol® PEG 860),polyglyceryl-10 stearate, polyglyceryl-10 laurate, polyglyceryl-10myristate, polyglyceryl-10 palmitate, polyglyceryl-10 linoleate,polyglyceryl-6 stearate, polyglyceryl-6 laurate, polyglyceryl-6myristate, polyglyceryl-6 palmitate, and polyglyceryl-6 linoleate.Polyglyceryl polyricinoleates (Polymuls) are also preferred surfactants.

Propylene Glycol Fatty Acid Esters

Esters of propylene glycol and fatty acids are suitable surfactants foruse in embodiments of the present invention. In this surfactant class,preferred hydrophobic surfactants include propylene glycol monolaurate(Lauroglycol FCC), propylene glycol ricinoleate (Propymuls), propyleneglycol monooleate (Myverol P-06), propylene glycol dicaprylate/dicaprate(Captex® 200), and propylene glycol dioctanoate (Captex® 800).

Sterol and Sterol Derivatives

Sterols and derivatives of sterols are suitable surfactants for use inembodiments of the present invention. Preferred derivatives include thepolyethylene glycol derivatives. A preferred surfactant in this class isPEG-24 cholesterol ether (Solulan C-24).

Polyethylene Glycol Sorbitan Fatty Acid Esters

A variety of PEG-sorbitan fatty acid esters are available and aresuitable for use as surfactants in embodiments of the present invention.Among the PEG-sorbitan fatty acid esters, preferred surfactants includePEG-20 sorbitan monolaurate (Tween-20), PEG-20 sorbitan monopalmitate(Tween-40), PEG-20 sorbitan monostearate (Tween-60), and PEG-20 sorbitanmonooleate (Tween-80).

Polyethylene Glycol Alkyl Ethers

Ethers of polyethylene glycol and alkyl alcohols are suitablesurfactants for use in embodiments of the present invention. Preferredethers include PEG-3 oleyl ether (Volpo 3) and PEG-4 lauryl ether (Brij30).

Sugar and its Derivatives

Sugar derivatives are suitable surfactants for use in embodiments of thepresent invention. Preferred surfactants in this class include sucrosemonopalmitate, sucrose monolaurate, decanoyl-N-methylglucamide,n-decyl-β-D-glucopyranoside, n-decyl-β-D-maltopyranoside,n-dodecyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside,heptanoyl-N-methylglucamide, n-heptyl-β-D-glucopyranoside,n-heptyl-β-D-thioglucoside, n-hexyl-β-D-glucopyranoside,nonanoyl-N-methylglucamide, n-nonyl-β-D-glucopyranoside,octanoyl-N-methylglucamide, n-octyl-β-D-glucopyranoside, andoctyl-β-D-thioglucopyranoside.

Polyethylene Glycol Alkyl Phenols

Several PEG-alkyl phenol surfactants are available, such as PEG-10-100nonyl phenol and PEG-15-100 octyl phenol ether, Tyloxapol, octoxynol,nonoxynol, and are suitable for use in embodiments of the presentinvention.

Polyoxyethylene-Polyoxypropylene (POE-POP) Block Copolymers

The POE-POP block copolymers are a unique class of polymericsurfactants. The unique structure of the surfactants, with hydrophilicPOE and hydrophobic POP moieties in well-defined ratios and positions,provides a wide variety of surfactants suitable for use in embodimentsof the present invention. These surfactants are available under varioustrade names, including Synperonic PE series (ICI); Pluronic® series(BASF), Emkalyx, Lutrol (BASF), Supronic, Monolan, Pluracare, andPlurodac. The generic term for these polymers is “poloxamer” (CAS9003-11-6). These polymers have the formula: HO(C2H4O)a(C3H6O)b(C2H4O)aHwhere “a” and “b” denote the number of polyoxyethylene andpolyoxypropylene units, respectively.

Preferred hydrophilic surfactants of this class include Poloxamers 108,188, 217, 238, 288, 338, and 407. Preferred hydrophobic surfactants inthis class include Poloxamers 124, 182, 183, 212, 331, and 335.

Sorbitan Fatty Acid Esters

Sorbitan esters of fatty acids are suitable surfactants for use inembodiments of the present invention. Among these esters, preferredhydrophobic surfactants include sorbitan monolaurate (Arlacel 20),sorbitan monopalmitate (Span-40), sorbitan monooleate (Span-80),sorbitan monostearate.

The sorbitan monopalmitate, an amphiphilic derivative of Vitamin C(which has Vitamin C activity), can serve two important functions insolubilization systems. First, it possesses effective polar groups thatcan modulate the microenvironment. These polar groups are the samegroups that make vitamin C itself (ascorbic acid) one of the mostwater-soluble organic solid compounds available: ascorbic acid issoluble to about 30 wt/wt % in water (very close to the solubility ofsodium chloride, for example). And second, when the pH increases so asto convert a fraction of the ascorbyl palmitate to a more soluble salt,such as sodium ascorbyl palmitate.

Ionic Surfactants

Ionic surfactants, including cationic, anionic and zwitterionicsurfactants, are suitable hydrophilic surfactants for use in embodimentsof the present invention. Preferred ionic surfactants include quaternaryammonium salts, fatty acid salts and bile salts. Specifically, preferredionic surfactants include benzalkonium chloride, benzethonium chloride,cetylpyridinium chloride, docecyl trimethyl ammonium bromide, sodiumdocecylsulfates, dialkyl methylbenzyl ammonium chloride, edrophoniumchloride, domiphen bromide, dialkylesters of sodium sulfonsuccinic acid,sodium dioctyl sulfosuccinate, sodium cholate, and sodium taurocholate.These quaternary ammonium salts are preferred additives. They can bedissolved in both organic solvents (such as ethanol, acetone, andtoluene) and water. This is especially useful for medical devicecoatings because it simplifies the preparation and coating process andhas good adhesive properties. Water insoluble drugs are commonlydissolved in organic solvents.

Fat-Soluble Vitamins and Salts Thereof

Vitamins A, D, E and K in many of their various forms and provitaminforms are considered as fat-soluble vitamins and in addition to these anumber of other vitamins and vitamin sources or close relatives are alsofat-soluble and have polar groups, and relatively high octanol-waterpartition coefficients. Clearly, the general class of such compounds hasa history of safe use and high benefit to risk ratio, making them usefulas additives in embodiments of the present invention.

The following examples of fat-soluble vitamin derivatives and/or sourcesare also useful as additives: Alpha-tocopherol, beta-tocopherol,gamma-tocopherol, delta-tocopherol, tocopherol acetate, ergosterol,1-alpha-hydroxycholecal-ciferol, vitamin D2, vitamin D3, alpha-carotene,beta-carotene, gamma-carotene, vitamin A, fursultiamine,methylolriboflavin, octotiamine, prosultiamine, riboflavine, vintiamol,dihydrovitamin K1, menadiol diacetate, menadiol dibutyrate, menadioldisulfate, menadiol, vitamin K1, vitamin K1 oxide, vitamins K2, andvitamin K—S(II). Folic acid is also of this type, and although it iswater-soluble at physiological pH, it can be formulated in the free acidform. Other derivatives of fat-soluble vitamins useful in embodiments ofthe present invention may easily be obtained via well known chemicalreactions with hydrophilic molecules.

Water-Soluble Vitamins and their Amphiphilic Derivatives

Vitamins B, C, U, pantothenic acid, folic acid, and some of themenadione-related vitamins/provitamins in many of their various formsare considered water-soluble vitamins. These may also be conjugated orcomplexed with hydrophobic moieties or multivalent ions into amphiphilicforms having relatively high octanol-water partition coefficients andpolar groups. Again, such compounds can be of low toxicity and highbenefit to risk ratio, making them useful as additives in embodiments ofthe present invention. Salts of these can also be useful as additives inthe present invention. Examples of water-soluble vitamins andderivatives include, without limitation, acetiamine, benfotiamine,pantothenic acid, cetotiamine, cyclothiamine, dexpanthenol, niacinamide,nicotinic acid, pyridoxal 5-phosphate, nicotinamide ascorbate,riboflavin, riboflavin phosphate, thiamine, folic acid, menadioldiphosphate, menadione sodium bisulfite, menadoxime, vitamin B12,vitamin K5, vitamin K6, vitamin K6, and vitamin U. Also, as mentionedabove, folic acid is, over a wide pH range including physiological pH,water-soluble, as a salt.

Compounds in which an amino or other basic group is present can easilybe modified by simple acid-base reaction with a hydrophobicgroup-containing acid such as a fatty acid (especially lauric, oleic,myristic, palmitic, stearic, or 2-ethylhexanoic acid), low-solubilityamino acid, benzoic acid, salicylic acid, or an acidic fat-solublevitamin (such as riboflavin). Other compounds might be obtained byreacting such an acid with another group on the vitamin such as ahydroxyl group to form a linkage such as an ester linkage, etc.Derivatives of a water-soluble vitamin containing an acidic group can begenerated in reactions with a hydrophobic group-containing reactant suchas stearylamine or riboflavin, for example, to create a compound that isuseful in embodiments of the present invention. The linkage of apalmitate chain to vitamin C yields ascorbyl palmitate.

Amino Acids and Their Salts

Alanine, arginine, asparagines, aspartic acid, cysteine, cystine,glutamic acid, glutamine, glycine, histidine, proline, isoleucine,leucine, lysine, methionine, phenylalanine, serine, threonine,tryptophan, tyrosine, valine, and their derivatives are other usefuladditives in embodiments of the invention.

Certain amino acids, in their zwitterionic form and/or in a salt formwith a monovalent or multivalent ion, have polar groups, relatively highoctanol-water partition coefficients, and are useful in embodiments ofthe present invention. In the context of the present disclosure we take“low-solubility amino acid” to mean an amino acid which has solubilityin unbuffered water of less than about 4% (40 mg/ml). These includecystine, tyrosine, tryptophan, leucine, isoleucine, phenylalanine,asparagine, aspartic acid, glutamic acid, and methionine.

Organic Acids and Their Esters and Anhydrides

Examples are acetic acid and anhydride, benzoic acid and anhydride,acetylsalicylic acid, diflunisal, 2-hydroxyethyl salicylate,diethylenetriaminepentaacetic acid dianhydride,ethylenediaminetetraacetic dianhydride, maleic acid and anhydride,succinic acid and anhydride, diglycolic anhydride, glutaric anhydride,ascorbic acid, citric acid, tartaric acid, lactic acid, oxalic acidaspartic acid, nicotinic acid, 2-pyrrolidone-5-carboxylic acid, and2-pyrrolidone.

These esters and anhydrides are soluble in organic solvents such asethanol, acetone, methyl ethyl ketone, ethyl acetate. The waterinsoluble drugs can be dissolved in organic solvent with these estersand anhydrides, then coated easily on to the medical device, thenhydrolyzed under high pH conditions. The hydrolyzed anhydrides or estersare acids or alcohols, which are water soluble and can effectively carrythe drugs off the device into the vessel walls.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Aqueous Aerosol Formulation

An exemplary aqueous formulation of rapamycin was prepared using thefollowing components.

Component Amount (g) Mass Fraction (w/w) rapamycin 0.1  0.01% ethanol250   25% propylene glycol 250   25% polysorbate 80 0.02 0.002% water500   50% Total 1000

Blending Procedure: in a 1000 ml amber volumetric flask, blend 250propylene glycol with 250 ethanol until uniform. Then sequentiallydissolve first 100 mg rapamycin then 20 mg polysorbate 80 in thepropylene glycol and ethanol solution. Add water to bring the volumetricto 1000 ml and stir or sonicate until uniform and all the rapamycin isdissolved. Store at controlled temperature away from light.

Example 2: Dry Powder Formulation

Batch 06RP68.HQ00008 and 06RP68.HQ00009. These two formulations are eacha blend of micronized drug (rapamycin) particles dispersed onto thesurface of lactose carrier particles. The final composition of eachbatch comprises 1% (w/w) drug particles having a mean diameter of about2.60 microns and 3.00 microns, respectively. Drug particles having asuitable size range are made by wet polishing (06RP68.HQ00008) or jetmilling (06RP68.HQ00009), as described below. While this example used 1%(w/w) rapamycin, a range 0.5 to 20% is practicable. The carrierparticles consist of a blend of two carriers, Respitose® SV003, presentat 95.5% (w/w) and having particle sizes of about 30 to 100 microns(equivalent spherical diameter), and Respitose® LH300 (Lactohale 300)present at 5.5% (w/w) and having particle sizes less than 10 microns(equivalent spherical diameter). After blending, the blends were assayedto confirmed homogeneity and drug content of 1%.

To reduce drug particle agglomeration and aid in the aerosolization ofdrug particles several other excipients are optionally included.Optional excipients include phospholipids, such asdipalmitylphosphatidylcholine (DPPC) and lecithin, and metal fatty acidsalts, such as magnesium stearate. These can be coated on the carrierparticles in weight ratio of excipient to large carrier particle rangingfrom 0.01 to 0.5%.

Capsule Filling: 20 milligrams of the powder blends from Batch06RP68.HQ00008 and Batch 06RP68.HQ00009 were loaded into size #3 HPMCcapsules to produce drug product. For these blends it was feasible toload from 5 to 35 milligrams of drug into #3 size capsules and emptygreater than 95% of the loaded blend from the capsule upon actuation inPlastiape® RS01 Model 7 or Plastiape® RS00 Model 8 devices at flow ratesranging from 60 to 100 liters per minute.

Example 3: Determination of Rapamycin in Lung and Blood FollowingAdministration By Oropharyngeal Aspiration (OPA) and Oral Gavage toC57BL6 Mice

This study was conducted to evaluate the concentration of rapamycin inmale C57BL/6 mice after administration of rapamycin at a very hightarget dose of 1 mg/kg by gavage and oropharyngeal aspiration (OPA). Amethod for the analysis of rapamycin in mouse blood and lung homogenatewas developed using liquid chromatography with tandem mass spectrometrydetection (LC-MS/MS). Calibration curves of rapamycin using triplicateconcentrations were analyzed between 1 ng/mL and 2000 ng/mL in mouseblood, and between 2 ng/mL and 20,000 ng/mL in mouse lung homogenate.Accuracy, precision and linearity were within expected ranges.

In pilot studies, the efficiency of vehicle delivery to the lungs viaoropharyngeal aspiration with a volume of 50 μL per mouse was evaluatedby administration of Evans Blue dye. The presence of blue dye only inlungs was verified visually, and the absence of blue dye in the stomachdemonstrated that delivery to the stomach was avoided in the procedureused.

Rapamycin was administered to male C57BL/6 mice (N=6) by gavage at adose of 1.0 mg/kg either orally or via OPA. The oral dose was formulatedusing pharmaceutical oral liquid formulation Rapamune Oral® (Pfizer).Rapamycin for OPA was prepared by dissolving the test article in anappropriate volume of ethanol, and then addition of an appropriatevolume of water to prepare a 10% ethanol solution at a concentration of1 mg rapamycin/mL. Rapamycin was administered to 2 groups of 6 maleC57BL/6 mice by OPA under isoflurane anesthesia. An additional group of6 mice received vehicle only (10% ethanol in water). At 1 h afteradministration a group of 6 mice receiving oral and OPA rapamycin wereeuthanized, and blood was obtained by cardiac puncture, and the lungsremoved. The remaining mice in each group administered rapamycin orvehicle by OPA were observed for an additional 3 days. At the 72-hnecropsy, blood was obtained by cardiac puncture and the lungs removed.No adverse effects were observed in rapamycin- or vehicle-treated micein the 72 h period following dosing.

The concentration of rapamycin was determined in the collected blood andin lung homogenate by LC-MS/MS. At 1 h following OPA of rapamycin, theconcentration of rapamycin was ˜6 fold higher in lung tissue (3794±1259ng/g tissue) than in blood (641±220 ng/ml). Following oraladministration of a similar dose of rapamycin, the 1-h lung and bloodconcentrations of rapamycin were 71±43 ng/g and 23±16 ng/mL,respectively. Lung homogenate concentrations following OPA were 53-foldhigher than those measured following oral administration of the samehigh dose (1 mg/kg) of rapamycin. The data suggests that delivery oflower doses of rapamycin to lung (dose levels that do not saturatesystem) will result in rapamycin levels in the lung that can be achievedby oral dosing but with significantly less rapamycin in the blood thanoccurs with oral dosing.

Materials and Methods

Test Substance: Sirolimus (Rapamune, Rapamycin) MW 914.172, C₅₁N₇₉NO₁₂,CAS NUMBER: 53123-88-9. Source (for oral gavage): Rapamune Oral®(Pfizer) for oral administration, Lot No.: MWGT, Expiration: July 2016.Source (for OPA): Rapamycin (Sirolimus) solid, LC Laboratories, WoburnMass., Lot No.: ASW-127, Expiration: December 2023.

Animals: Male C57BL/6 mice, approximately 8 weeks of age, from CharlesRiver Laboratories, Inc, Raleigh, N.C. Animals were fed Certified PurinaRodent Chow #5002 and were furnished tap water ad libitum. The analysisof each feed batch for nutrient levels and possible contaminants wasperformed by the supplier, examined by the Study Director, andmaintained in the study records. The feed was stored at approximately60-70° F., and the period of use did not exceed six months from themilling date. Mice were housed (one per cage) in polycarbonate cageswith stainless steel bar lids accommodating a water bottle. Cage sizesare approximately 11.5″×7.5″×5″ high (70 sq. in. floor space) for mice.Contact bedding was Sani-Chips hardwood chips (P. J. Murphy ForestProducts Co.; Montville, N.J.). Mice were quarantined for a period of 5days before use on a study. A veterinarian or qualified designeeexamined the animals prior to their release from quarantine. Temperatureand relative humidity in RTI animal rooms were continuously monitored,controlled, and recorded using an automated system (Siebe/Barber-ColmanNetwork 8000 System with Revision 4.4.1 for Signal® software [SiebeEnvironmental Controls (SEC)/Barber-Colman Company; Loves Park, Ill.]).The target environmental ranges were 64-79° F. (18° C.-26° C.) fortemperature and 30-70% relative humidity, with a 12-h light cycle perday. At the end of the in-life phase, the mice were euthanized byoverexposure to carbon dioxide.

Test Chemical Preparation: Evans Blue was prepared at 0.5% w/v insterile distilled water. Rapamune Oral® was administered as supplied fororal dosing. Rapamycin (solid) was dissolved in ethanol and diluted withsterile distilled water to provide a final concentration of 0.5 mg/mLin10% ethanol.

Dosing: Each animal was weighed prior to dosing to determine the amountof dose to be administered. A single gavage dose was administered usinga 100-μL glass syringe (Hamilton, Reno, Nev.) fitted with a ball-tipped20-G stainless steel gavage dosing needle (Popper & Sons Inc., New HydePark, N.Y.). The dose administered to each animal was determined fromthe weight of the full syringe minus that of the empty syringe. Thedosing time was recorded. Dosing of animals was spaced apart to allowblood collection at the appropriate times. The dose formulationsadministered to each group are shown below.

For oropharyngeal aspiration group animals, a single dose of rapamycin(50 μL) was administered to each mouse under isoflurane anesthesia,using a 100 μL glass syringe (Hamilton, Reno, Nev.) fitted with aball-tipped 24-G stainless steel gavage dosing needle (Popper & SonsInc., New Hyde Park, N.Y.). The mouse was weighed prior to dosing, andthe dose of rapamycin administered was recorded by weight. Each mousewas anesthetized with isoflurane, and restrained with the mouth open.The tongue was held to one side of the mouth with forceps, and the dosewas slowly injected into the distal part of the oral cavity. Thenostrils were covered with a finger for two breaths to ensure aspiration(Rao et al., 2003).

TABLE 1 Study Design Summary Target Target Target Dose No. Dose DoseDose Collection Samples Group Route Compound Animals (mg/ml) (ul)(mg/kg) Time Collected 1 OA Evans 6 — 50 0 1 blood, Blue lung 2 OARapamycin 6 0.5 50 1.0 1 blood, lung 3 Gavage Rapamune 6 1.0 25 1.0 1blood, Oral lung 4 OA Vehicle 6 0 50 1.0 72 blood, lung 5 OA Rapamycin 60.5 50 1.0 72 blood, lung

Collection of Blood and Lung Samples: At study termination (1 or 72 hafter dosing), mice were anesthetized by exposure to CO2, and blood wascollected by cardiac puncture with dipotassium EDTA as anticoagulant.Lung tissue was excised and divided into the right and left lung. Theleft lung was used for analysis, and the right lung flash frozen inliquid nitrogen and stored at −70° C. for further analysis.

Analysis of Samples for Rapamycin by LC-MS/MS: An LC-MS/MS method foranalysis of rapamycin in lung and blood was prepared based on thepublished method of Wu et al. (2012). The volumes of blood and lunghomogenate were reduced substantially from the published method.Triamcinolone was used as internal standard.

Lung homogenate was prepared by homogenization of weighed lung sampleswith 2.8-mm ball bearings in a homogenizer with tissue+deionized water(1:3 w/v) in a SPEX SamplePrep 2010 Geno/Grinder.

The concentrations of standards were arranged so that each standard camefrom an alternate stock standard. A six-point calibration curve, eachmade in triplicate, was employed for analyte quantitation. A simplelinear regression model with or without weighting was employed for curvefitting. The concentration range determined was from 1-2000 ng/mL inblood and 2-2000 ng/mL in lung homogenate.

The following method performance parameters were considered acceptable;the coefficient of determination, r2, of ≥0.98 forconcentration-response relationship; an accuracy of ≤±15% (forconcentrations above LOQ) or ≤±20% (for concentration at LOQ) of thenominal value. r² was greater than 0.999 in all analysis.

Thirty (30) μL of matrix, 30 μL of spiking solution (methanol for blanksand samples), 10 μL Internal standard solution (in MeOH) and 90 μL ofMeOH were pipetted into microcentrifuge tubes, vortexed briefly, thencentrifuged for 6 min at 10,000 RPM at ˜4° C. Aliquots (90 μL) ofsupernatant were transferred to LC vial inserts, and then analyzed byLC-MS/MS (Table 2).

TABLE 2 LC-MS/MS Method Column Waters Acquity UPLC HSS T3 1.8 μm, 2.1 ×50 mm with VanGuard 2.1 × 5 mm HSS T3 1.8 μm. Mobile Phase A 10 mMAmmonium Acetate in water, 0.1% acetic acid Mobile Phase B MeOHInjection Vol 2 ul Flow Rate 0.5 ml/min Gradient 70% A for 1 min, alinear gradient to 5% A from 1-3 min, held for 1 min, a linear gradientto 70% A from 4-5.1 min, and held at 70% until 6 min Rapamycin MRM931.70→864.70 Triamcinolone 395.30→357.20 (IS MRM)

Data Collection and Reporting: Study data was collected and reported inthe Debra™ system version 5.5.10.72 (Lablogic Systems Ltd., Sheffield,England). This includes data for animal body weights, dose administered,dose time, and sample collection times. Calculations of doseadministered and sample collection times were reported with the Debra™system.

Results

Rapamycin Analysis: The analysis of rapamycin was set up of samplevolumes of 30 μL of blood and lung homogenate. Example chromatograms areshown for rapamycin and internal standard in blood and lung (FIGS. 1A-Band 2A-B, respectively). Prior to the generation of study samples,triplicate calibration curves were generated for lung and blood, toverify method performance. The calibration range was from 1.0-2000 ng/mLfor blood and 1-20,000 ng/mL for lung homogenate. Lung homogenate wasprepared with 1 g of lung tissue homogenized in 3 volumes of water, toyield a 1:4 homogenate. Calibration curves are shown in FIG. 3 and FIG.4 for blood, lung homogenate, respectively, and solvent.

Oropharyngeal Aspiration: Prior to the administration of rapamycin byoropharyngeal aspiration, administration of Evans Blue was used toverify that the OPA delivered the dose to the lungs. Mice wereanaesthetized with isoflurane and administered Evans Blue by OPA, usinga syringe equipped with a blunt needle. Immediately following OPA, themice were euthanized and the lungs and stomach examined visually toensure that the Evans Blue dye was delivered to the lungs, and was notdelivered to the stomach. Four mice were successfully administered EvansBlue with all of the dye appearing to be located in the lungs and nonein the stomach.

Rapamycin Administration: The weight of dose solution administered wasdetermined by weighing the charged syringe with dose solution prior todosing, and weighing following dosing. The weight of dose solutionadministered was used to calculate the amount of rapamycin administered.The time of dosing was recorded as 0. Animals in groups 2 and 3 wereeuthanized at 1 h after dosing. Animals in groups 4 and 5 were observedfor 72 h after dosing. No significant clinical signs were observed inany of the groups.

Rapamycin Analysis in Blood and Lung: Rapamycin was analyzed in mouseblood and left lung homogenate in all of the samples collected (FIGS.5A-B and 6A-B, respectively). Samples of the right lung from each animalwere saved for potential further analysis. Summary data for the samplesare provided in Table 3.

TABLE 3 Concentration of Rapamycin in Blood and Lung Following Oral andOropharyngeal (OPA) Administration of Rapamycin to Mice (1 mg/kg) AnimalRoute of Time Lung (ng/g Blood No. Admin post-dose (h) tissue) (ng/ml)2-07 OPA 1 5040 615.5 2-08 OPA 1 2642 455.5 2-09 OPA 1 4500 622 2-10 OPA1 1874 364.5 2-11 OPA 1 4006 937 2-12 OPA 1 4700 848.5 Mean 3794 641 SD1259 220 3-13 Gavage 1 109.8 49.85 3-14 Gavage 1 24.66 11.3 3-15 Gavage1 122.8 28.7 3-16 Gavage 1 54 28.35 3-17 Gavage 1 <LOQ 2.845 3-18 Gavage1 43 19.35 Mean 71 23 SD 43 16 5-25 OPA 72 11.7 <ELOQ 5-26 OPA 72 11.4<ELOQ 5-27 OPA 72 15.7 <ELOQ 5-28 OPA 72 10.8 <ELOQ 5-29 OPA 72 11.9<ELOQ 5-30 OPA 72 13.6 <ELOQ Mean 12.5 SD 1.9

For all sample sets, a triplicate calibration curve was analyzed withthe sequence of standard set, sample replicate 1, standard set, samplereplicate sample 2, standard set. At 1 h following OPA of rapamycin, theconcentration of rapamycin was ˜6 fold higher in lung tissue (3794±1259ng/g tissue) than in blood (641±220 ng/ml). Following oraladministration of a similar dose of rapamycin, the 1-h lung and bloodconcentrations of rapamycin were 71±43 ng/g and 23±16 ng/mL,respectively. Lung homogenate concentrations following OPA were 53-foldhigher than those measured following oral administration of same highdose (1 mg/kg) of rapamycin.

Discussion

This study investigated the concentration of rapamycin in blood and lungtissue following administration of rapamycin by gavage in a commercialoral formulation, and by oropharyngeal administration (OPA) as asuspension prepared in 10% aqueous ethanol. No adverse effects wereobserved in rapamycin- or vehicle-treated mice up to 72 h followingdosing via OPA. Prior to administration of rapamycin, an analyticalmethod was developed, and the administration of a dye into the lung byOPA was verified. The concentrations of rapamycin in lung following OPAwere 6-fold higher than in blood. At 72 h after OPA, rapamycin was belowthe limit of quantitation in blood, but was detectable in lung. Thisstudy indicated that rapamycin is available systemically followingpulmonary administration, and that lung tissue concentrations greatlyexceed that of blood at early and late time points following delivery tothe lung.

These results further demonstrate that rapamycin delivered directly tothe lung achieves an unexpectedly high local concentration of drug inlung tissue compared to the blood. This result was entirely unexpectedfrom what is known about the pharmacology of rapamycin, which predictsan approximately equal concentration of the drug in lung tissue and theblood because rapamycin is known to distribute evenly throughout bodilytissues and should be cleared rapidly from the lung due to its highlipophilicity. Accordingly, these results indicate that directadministration of rapamycin to the lungs should be able to achieve ahigh enough delivered dose for therapeutic efficacy while at the sametime achieving almost undetectable systemic availability, therebyeliminating the toxicities associated with oral administration that aredue to systemic exposure to the drug. While toxicity to the lung itselfis also of concern in view of earlier studies, the results here furtherunexpectedly indicate that relatively high amounts of rapamycin were notacutely toxic to lung tissue.

Example 4: Rapamycin Inhibits the Viability of Tsc2 Mutant Cells andInhibits S6 Phosphorylation

The anti-proliferative activity of rapamycin was tested against theangiomyolipoma (AML) derived TSC2 deficient TRI-AML101 cell line. TheTRI-AML101 cell line was derived from a TSC2 deficient primary human AMLprovided by Dr. Elizabeth Henske (Fox Chase Cancer Center, Philadelphia,Pa.). The tumor cells were immortalized by a two step process. First thecells were infected with the amphotropic retrovirus LXSN16E6E7 thatencodes the HPV16 E6 and E7 open reading frames and neomycin resistancecassette. Cells were expanded and neomycin-selected. Individual cloneswere isolated and frozen down. Next, the human Telomerase gene (hTERT)with hygromycin resistance cassette (pLXSN hTERT-hyg plasmid) wastransfected in and a stable line was selected by hygromycin selection.

The activity of rapamycin was tested on the TRI-AML101 cells byperforming 10 point dose response analysis of cell viability. Twothousand cells in 50 uL of growth media (DMEM, 10% FBS, and 1%Penicillin/Streptomycin) were plated per well in a 96 well plate. 24hours after plating cells another 50 uL of growth medium containingrapamycin (0.0005-5000 nM, 10-fold dilutions, 0.1% final DMSOconcentration) or DMSO only was added to the cells. 72 hours aftercompound addition, relative cell viability was determined byCellTiter-Glo® luminescence assay (Promega) and expressed as apercentage relative to vehicle (DMSO) treated control cells. Rapamycininhibited viability at concentrations as low as 0.05 nM (FIG. 7B).Inhibition of the mTOR pathway was also demonstrated by measuring thelevels of phosphorylated S6 by western blot. AML cells were incubatedwith 20 nM rapapycin for 24 hours. Western blot analysis was thenperformed and demonstrated that rapamycin potently inhibits S6phosphorylation (FIG. 7A).

Example 5: S6 Phosphorylation in Mouse Lung Following Oral and OPAAdministration of Rapamycin

As discussed above, our experiments showing the tissue distribution ofrapamycin in lung and blood following oral administration and OPAdemonstrated that direct administration of rapamycin to the lungs shouldbe able to achieve a high enough delivered dose for therapeutic efficacywhile at the same time achieving very low systemic exposure to the drug,thereby simultaneously improving therapeutic efficacy and eliminatingmany of the toxicities associated with oral administration of rapamycin.To validate this approach, we used the presence of phosphorylated S6protein in murine lung tissue as a biomarker for mTOR activity. In themouse strain used (C57bl/6), the mouse airway and alveolar epithelialcells have constitutively active (phosphorylated, “p”) S6 protein. TheS6 protein is typically phosphorylated by S6K which is downstream ofmTORC1 and is activated, for example downstream of growth factors suchas epidermal growth factor (EGF), AKT, ERK, and RSK. mTORC1 promotescell growth and proliferation by stimulating anabolic processes such asbiosynthesis of lipids, proteins, and organelles, and suppressingcatabolic processes such as autophagy. The mTORC1 pathway senses andintegrates intracellular and extracellular signals, including growthfactors, oxygen, amino acids, and energy status, in order to regulate awide range of processes, such as protein and lipid synthesis andautophagy. mTORC1 is acutely sensitive to rapamycin.

In the present study, lung tissue was taken from the C57bl/6 micetreated as discussed above, either with vehicle (n=6), or 1 mg/kgrapamycin administered via OPA (n=6) or via oral gavage (n=6) at twotime points post dosing, 1 hr and 72 hours. As discussed above,following OPA at 1 hr, rapamycin was detected at 641 ng/ml in the bloodand 3794 ng/g tissue in the lung, and at 72 hrs was still detectable inthe lung at 12.5 ng/g while being undetectable in the blood at that timepoint. Conversely, following oral (gavage) administration, at 1 hr,rapamycin was detected at 23 ng/ml in the blood and 71 ng/g tissue inthe lung, and at 72 hrs was undetectable in either the lung or blood. Asshown by the data in FIG. 8A, the level of phosphorylated S6 (pS6) wasreduced substantially by both OPA and orally administered rapamycin at 1hr and remained suppressed at 72 hr for OPA. pS6 was highest in thevehicle control because these mice have constitutively active mTORsignaling. These data show that a delivered dose of rapamycin sufficientto achieve about 70 ng/g drug in the lung substantially abrogates mTORsignaling in the lung tissue as measured by pS6 protein and that mTORsignaling remains suppressed at levels as low as 12.5 ng/g. Theseresults validate our approach to utilize inhaled rapamycin for thetreatment of diseases and disorders characterized at least in part byaberrantly high mTOR pathway activity, by demonstrating that inhaledrapamycin can be delivered at much lower doses than orally administeredrapamycin to simultaneously achieve high therapeutic efficacy and verylow toxicity.

Example 6: Inhaled Rapamycin Inhibits S6 Phosphorylation in Lung Tissue

Normal Sprague-Dawley Rats were dosed by inhalation to achieve targetdose of 0.354 mg/kg of rapamycin (LAM-001) (N=36) and subgroups of 6animals were sacrificed at the following time points (1) Predose, (2)Midway dosing, (3) Immediate post dosing, (4) 2 hours post dosing, (5) 4hours post dosing and (6) 12 hours post dosing on Study Day 1. CharlesRiver determined the lung concentration of rapamycin for each sacrificedanimal at their subgroup time point, the average rapamycin concentrationin nanograms rapamycin per gram of tissue (ng/g) for each group isreported in the table below.

TABLE 4 Rapamycin in lung tissue following administration by inhalationTarget dose Midway Immediate 2 hr post- 4 hr post- 12 hr post- 11 ug Day1 Dosing post-dosing dosing dosing dosing Avg Lung 462 560 250 192 95rapa (ng/g):

Lungs samples from each animal were collected and snap frozen. Theindividual frozen lung samples were homogenized (Qiagen TissueLyser LTaccording to manufacturer's protocols) in 1× RIPA Buffer with proteaseand phosphatase inhibitors. The lung homogenates were analyzed bywestern blot analysis of the mTOR downstream target, Phospho-S6Ribosomal Protein (Ser240/244) (Cell Signaling Technology antibody,clone D68F8) as compared to total S6 protein S6 Ribosomal Protein (CellSignaling Technology antibody, clone 5G10) levels. The Western blotimages were analyzed by NIH imageJ v1.48 to generate the respectiveantibody reactivity/intensity and create the ratio of S6 phosphorylation(S6-P) to total S6 intensities for each lung sample. The S6-P/total S6ratios (y-axis) for sample organized by timepoint group (X-axis) wereplotted on a one grouping variable scatter plot vertical graph(GraphPad, version 4.0), all samples in groups are represented by filledin black dots (•) on the graph and average is shown by the horizontalline between the dots within each respective group (FIG. 8B).

Example 7: Inhaled Rapamycin Shows Unexpected Biodistribution to theLung

It had been reported in the literature that rapamycin collects in thelungs after high oral or IV doses (Yanez, J. et. al., Pharmacometricsand Delivery of Novel Nanoformulated PEG-b-poly(ε-caprolactone) Micellesof Rapamycin, Cancer Chemotherapy and Pharmacology, 61 (1), 133-1442007). A study reported that after administering a single dose of 10.0milligrams/kilogram (mg/kg) to Spraque-Dawley (SD) rats, the amount ofrapamycin in the lungs after allowing time for distribution through thetissue compartments (24 hours) was 721 nanograms/gram (ng/g),approximately 19 times the concentration in the blood (Table 5).

TABLE 5 Biodistribution of Rapamycin in the lung and blood after IVadministration per Yanez et al. Yanez 1 Day (24 hours after single dose)IV Dose Lung/ Daily Human Rapamycin (mg/kg/ Rapamycin in Blood DoseEquiv in Blood day) Lung (ng/g) Ratio (mg) (ng/ml) 10 721 19 600 31

In an earlier separate study by Napoli (Napoli, K., et. al.,Distribution of Sirolimus in Rat Tissue, Clinical Biochemistry,30(2):135-142, 1997) a range of rapamycin doses to SD rats wereadministered daily, by oral and intravenous (IV) administration routes.After 14 days of IV administration rapamycin concentrations in lungtissue ranged, dose proportionally, from a 200 to 900 ng/g,approximately 23 to 44 times the higher than the concentrations in theblood. But for oral administration much lower levels of rapamycinaccumulated in the lung for the same doses, even though the ratios oflung to blood concentrations of rapamycin were approximately the same(Table 6).

TABLE 6 Biodistribution of Rapamycin 24 hrs after the 14^(th) once dailyIV administration ON 14 days per Napoli et al. Napoli 14 Day QD (24hours after 14th dose) IV Dose Rapamycin Lung/ Daily Human Rapamycin(mg/kg/ in Lung Blood Dose Equivalent in Blood day) (ng/g) Ratio (mg)(ng/ml) 0.04 219 23 2.4 9.4 0.08 457 30 4.8 15.0 0.16 677 33 9.6 20.60.40 868 44 24 19.6 Rapamycin Rapamycin Oral Dose in Lung/ Daily Humanin (mg/kg/ Lung Blood Dose Equiv. Blood day) (ng/g) Ratio (mg) (ng/ml)0.4 42 53 3.4 0.8 0.8 59 28 6.7 2.1 1.6 175 24 13.4 7.4

When we examined the biodistribution of rapamycin followingadministration via inhalation, we found that rapamycin accumulates muchhigher in the lungs than would have been predicted based upon Napoli andYanez, even while the lung to blood ratios were similar. In a firststudy, rapamycin was administered to SD rats by inhalation at two dosesof (1) 1.0 mg/kg/day and (2) 0.0360 mg/kg/day, for a single day. Afterallowing 12 hours for distribution to tissue compartments, the rapamycintrough concentrations in the lungs for the high dose was about 14,800ng/g and the concentration of rapamycin in the lungs was approximately23 times higher than in the blood (Table 7). For the low dose, theconcentration in the lungs was 24 times higher than the concentration inthe blood (Table 7). Table 8 shows the trough lung concentration, themaximum and trough blood concentrations after repeated, once a daydosing for 5 days at the same two doses used in the previous experiment,i.e., 1.0 mg/kg/day and 0.0360 mg/kg/day.

TABLE 7 Biodistribution of Rapamycin via inhalation 12 hrs after asingle dose Daily Inhaled Human Dose Rapamycin Lung/ Dose Rapamycin(mg/kg/ in Lung Blood Equivavent in Blood day) (ng/g) Ratio (mg) (ng/ml)1.00 14831 23 60 645 0.0360 95 24 2.16 4

TABLE 8 Biodistribution of Rapamycin via inhalation once a day (measuredat trough day 5) Daily Human Inhaled Rapamycin Lung/ Dose Rapamycin Dosein Lung Blood Equivalent in Blood (mg/kg/day) (ng/g) Ratio (mg) (ng/ml)1.0000 17163 23 60 746 0.0360 87 22 2.16 4

The results of this initial study indicate that delivery of rapamycin tothe lungs by inhalation produced markedly higher concentrations of drugin the lung tissue than could be achieved by alternative routes ofadministration, e.g. oral or intravenous, according to the previous workof Yanez and Napoli. Moreover, the high amounts of rapamycin in the lungfollowing delivery via inhalation were unexpectedly higher based uponwhat would have been predicted from Yanez and Napoli. As bothintravenous and inhaled routes of administration have highbioavailability of rapamycin, the inhaled 1 mg/kg dose would have beenpredicted to achieve lung concentrations about 2.5 times those observedby Napoli's 0.4 mg/kg/day IV dose. Instead, the levels of rapamycin inthe lung were approximately 17 times higher when administered viainhalation (compare Table 7, 1 mg/kg/day via inhalation produced 14,831ng/g drug in lung versus Table 6 (Napoli), 0.40 mg/kg/day IV produced868 ng/g lung; 14,831/868=17). Similarly the 10 mg/kg intravenous doseadministered by Yanez would have been predicted to achieve a lungconcentration of rapamycin about 10 times higher than that achieved bythe 1 mg/kg inhaled dose. Instead, the intravenous dose achieved lungconcentrations approximately 20 times less than the inhaled dose(compare Table 7, 1 mg/kg/day via inhalation produced 14,831 ng/g drugin lung versus Table 5 (Yanez), 10 mg/kg/day IV produced 721 ng/g inlung; 14,831/721=21). This could possibly be due to low metabolicactivity in the lungs and slow passive or active transport of rapamycinfrom the lung tissue compartment into systemic circulation. Regardlessof the precise mechanism, these results indicate that delivery ofrapamycin to the lungs results in persistently high localconcentrations, while circulatory concentrations remain low.

The results of this initial study were replicated and expanded inadditional rat studies and dog studies. These subsequent studies werestructured to determine the repeat dose toxicity and toxicokinetics of adry powder aerosol formulation of 1% (w/w) rapamycin blended withlactose, administered by inhalation to normal Sprague-Dawley (SD) ratsand Beagle dogs. In the first study, standard cylindrical flow-throughnose-only inhalation chambers were utilized to administer the dry powderformulation to SD rats. For five consecutive days, animals weresubjected to test article for 300 minutes each day to achieve a targetdose of 0.354 mg/kg of rapamycin. Two sets of animals were used toperform the toxicokinetic measurements for this study. The first set ofanimals were dosed for 300 minutes on Study Day 1 and blood samples(N=36) and lung samples (N=36) were taken from animals were sacrificedin subgroups of 6 at the following time points: (1) Predose, (2) Midwaydosing, (3) Immediate post dosing, (4) 2 hours post dosing, (5) 4 hourspost dosing and (6) 12 hours post dosing. The second set of animals weredosed for 300 minutes for 5 consecutive days, and on Study Day 5, bloodsamples (N=36) and lung samples (N=36) were taken from animals weresacrificed in subgroups of 6 at the following time points (1) Predose,(2) Midway dosing, (3) Immediate post dosing, (4) 2 hours post dosing,(5) 4 hours post dosing and (6) 12 hours post dosing. The maximumconcentration of rapamycin in whole blood (ng/ml) and lung tissuesamples (ng/g), as well as the concentration of rapamycin 12 hourspost-dose.

A second repeat exposure study was conducted to evaluate the repeat dosetoxicity and toxicokinetics of the dry powder formulation administeredto SD rats and Beagle dogs for 28 days.

For the rat study, standard cylindrical flow-through nose-onlyinhalation chambers were utilized, as above. For 28 consecutive days,animals were exposed to test article for 300 minutes each day to achievea target doses of 0.167, 4.75 and 9.50 mg/kg of rapamycin respectively.For each of the three dosing groups, one set of animals (N=36) weredosed for 300 minutes each day for 28 consecutive days. On Study Days 1and 28, blood samples were taken from animals in subgroups of 6 at thefollowing time points: (1) Predose, (2) Midway dosing, (3) Immediatepost dosing, (4) 2 hours post dosing, (5) 4 hours post dosing, (6) 12hours post dosing and (7) 24 hours post dosing.

For the dog study, a positive flow delivery system (PFDS) consisting ofa central plenum and delivery arms was utilized. The central plenum wasof modular design with separate ports into which were attached 5delivery arms fitted with oronasal exposure masks fitted with inlet andoutlet tubes. The mask was fitted over the dog's muzzle in such a waythat the nose was inside the mask, allowing entrance and exit of air.During exposure, animals wore a harness and were placed on a restraintplatform. The harness was attached to two side poles on the platform inorder to restrict lateral movement of the dog. The front part of theharness was loosely attached to a hook on the front of the platform toprevent the animal from turning around. Dogs were exposed to testarticle for 60 minutes each day to achieve a target doses of 0.020 and0.053 mg/kg of rapamycin respectively. For each dosing group, one set ofanimals (N=6) was dosed for 60 minutes each day for 28 consecutive days.On Study Days 1 and 28, blood samples were taken from animals insubgroups of 6 at the following time points: (1) Predose, (2) postdosing (T=0), (3) 1 hour post dosing, (4) 4 hours post dosing, (5) 8hours post dosing, (6) 12 hours post dosing and (7) 24 hours postdosing. On Day 29, the dogs were sacrificed and a portion of their lungtissue was removed and minced for analysis of rapamycin content.

The maximum concentration of rapamycin in whole blood (ng/ml) and thetrough concentration of rapamycin are presented below along withextrapolations to human dosing. Also included in this table are thetrough levels of rapamycin in dog lung (ng/g) after 28-days of repeatdosing.

TABLE 9 Inhaled rapamycin rat data and extrapolations for human dosingRepeat Max Blood Lung 24 hr Emitted inhaled blood trough trough Dosedaily dose levels levels levels (ug) (ug) (ng/ml) (ng/ml) (ng/g) Rat* 505 7.6 2 N/A 1425 143 70.5 23.3 N/A 2850 285 77.8 21.0 N/A Dog** 160 407.4 1.9 32 424 106 27.3 5.9 61 Human 87 35 0.65 0.15 2 175 70 1.3 0.3 4*rat 28-day repeat daily dose study 7300225-blood data are average ofD28 **dog 28-day repeat daily dose study 7300227-blood and lung data areaverage of D28 **blood and lung estimates are extrapolated from the7300225 and 7300227 study results

Notably, based upon the results presented here, a therapeuticallyeffective dose of rapamycin in the lung in the range of about 5 ng/g inhumans could be achieved by administering less than about 100 microgramsto the lungs by inhalation. In contrast, achieving a comparable lungconcentration by oral delivery according to Yanez would require 4 to 16milligrams. To achieve a comparable lung concentration by IV deliveryaccording to Napoli, 60 to 600 micrograms would be required.

In addition, based upon the results presented here, the therapeuticrange of about 5 ng/g in the lung could be achieved with a lung to bloodpartitioning ratio of 13:1 when rapamycin is delivered via inhalation.This means that while rapamycin is within the therapeutic range in thelung tissue, maximum concentrations of only 650 to 1500 picograms/mlrapamycin would circulate in the blood. This low systemic exposure torapamycin is expected to reduce the toxicities and adverse drug eventsassociated with the much higher systemic exposure to rapamycin resultingfrom the higher levels of dosing required with oral or IVadministration.

In summary, the results described here demonstrate that administeringrapamycin to the lungs via inhalation advantageously provides for a lowdose of rapamycin required to achieve a therapeutically effective dosein the lung in the range of about 5 ng/g, combined with low systemicexposure to the drug, resulting in markedly improved therapeutic indexfor rapamycin.

Example 8: Size Reduction of Rapamycin for Inhalable Compositions

Particle size of rapamycin was reduced to a target range of 2.0μm<Dv50<3.0 μm using either a wet polishing or jet milling process. Forjet milling, a lab scale MCOne unit from Jetpharma was used with thefollowing operating conditions: venturi pressure 2-4 bar, millingpressure 3-5 bar, feed rate 90 g/h. For wet polishing, feed suspensionswere prepared using purified water. A microfluidics high pressurehomogenizer was used for the size reduction step and the resultingsuspension was spray-dried. Details of the wet polishing process are setforth below.

The high pressure homogenizer used for the size reduction step of thewet polishing process was a pilot-scale Microfluidics High PressureHomogenizer equipped with an auxiliary processing module (200 micron)and a 100 micron interaction chamber was used. The unit was operated at˜455 bar (˜30 bar in the intensifier module hydraulic pressure). Aftermicrofluidization the fluid was removed by spray drying to generate adry powder. A laboratory scale spray dryer, SD45 (BUCHI, model B-290Advanced) was equipped with a two fluid nozzle (cap and diameter were1.4 and 0.7 mm, respectively). Two cyclones in series were used (beingthe first the standard Buchi cyclone and the second the high-performanceBuchi cyclone) to collect the dried product. The spray drying unit wasoperated with nitrogen and in single pass mode, i.e. withoutrecirculation of the drying nitrogen. The aspirator, blowing nitrogen,was set at 100% of its capacity (flow rate at maximum capacity isapproximately 40 kg/h). The flow rate of the atomization nitrogen wasadjusted to a value in the rotameter of 40±5 mm. Before feeding theproduct suspension, the spray dryer was stabilized with purified water,during which the flow rate was adjusted to 6 ml/min (20% in theperistaltic pump). The inlet temperature was adjusted to achieve thetarget outlet temperature (45° C.). After stabilization of thetemperatures, the feed of the spray dryer was commuted from purifiedwater to the product suspension (keeping the same flow rate used duringstabilization) and the inlet temperature once again adjusted in order toachieve the target outlet temperature. At the end of the stocksuspension, the feed was once more commuted to purified water in orderto rinse the feed line and perform a controlled shut down. The dryproduct in the collection flasks under both cyclones was weighed and theyield calculated as the mass percentage of the dry product in relationto the total solids in the suspension fed to the high pressurehomogenizer.

Particle size distribution was analyzed by laser diffraction. Solidstate characterization (for polymorphic form and purity) was performedby high pressure liquid chromatography (HPLC), X-ray powder diffraction(XRPD), and differential scanning calorimetry (mDSC). Water content wasdetermined by the Karl Fischer method.

Jet milling produced crystalline rapamycin powder with a monodisperseparticle size distribution having Dv10 of 1.5 microns, a DV50 of 2.7microns and a Dv90 of 4.9 microns, as shown in Table 10 below.

Wet polishing produced crystalline rapamycin powder with a monodisperseparticle size distribution having a Dv10 of 1.0 microns, a Dv50 of 2.4microns and a Dv90 of 5.0 microns (Table 11).

Both methods produced particles of rapamycin within the target range andneither process showed an impact on polymorphic form or purity of therapamycin. The tables below show in-process control data for the jetmilling and wet polishing processes. The data indicate that bothprocesses were able to produce API particle sizes within the targetrange without impacting API purity or polymorphic form.

TABLE 10 Jet Milling Data Dv10 Dv50 Dv90 PSD μm 1.51 2.74 4.91 XRPD —Similar to API (sirolimus) diffractogram mDSC (T_onset, ° C.) 182.2 KF %w/w 0.30 HPLC Assay (% w/w) 99.5 (% area) Sirolimus 99.35

TABLE 11 Wet Polishing Data Dv10 Dv50 Dv90 PSD μm 1.05 2.42 4.97 XRPD —Similar to API (sirolimus) diffractogram mDSC (T_onset, ° C.) 185.7 KF %w/w 0.30 HPLC Assay (% w/w) 99.0 (% area) Sirolimus 99.42

Example 9: Aerosol Performance Testing of Dry Powder Compositions

The capsules produced in the example above were placed into the deviceindicated in the tables below and actuated. The aerosol performancedelivered from the devices/capsules containing blends from Batch06RP68.HQ00008 and Batch 06RP68.HQ00009 were characterized using a nextgeneration impactor (NGI) according to the methods described in Chapters905 and 601 of the USP. The aerosols were tested at flow rates of 60 and100 liters per minute (LPM). The fine particle dose (FPD) and fineparticle fraction (FPF) are shown in the tables below. Mass medianaerodynamic diameters (MMAD) and geometric standard deviations (GSD) arealso shown.

TABLE 12 06RP68.HQ00008 (Wet Polished) + Plasitape RS01 Model 7 60 LPM100 LPM Mean % RSD Mean % RSD FPD (μg) 57.31 2.37 67.21 12.46 FPF (%)39.49 1.85 44.12 8.99 MMAD (μm) 2.81 2.22 2.49 11.97 GSD 2.02 0.99 2.198.25

TABLE 13 06RP68.HQ00008 (Wet Polished) + Plastiape RS00 Model 8 60 LPM100 LPM Mean % RSD Mean % RSD FPD (μg) 58.40 0.98 62.39 6.35 FPF (%)39.68 1.68 41.34 3.70 MMAD (μm) 2.63 7.28 2.58 6.00 GSD 2.05 3.69 2.156.32

TABLE 14 06RP68.HQ00009 (Jet Milled) + Plastiape RS01 Model 7 60 LPM 100LPM Mean % RSD Mean % RSD FPD (μg) 52.33 6.72 58.51 15.84 FPF (%) 33.733.91 36.69 9.86 MMAD (μm) 3.32 2.27 3.02 4.14 GSD 2.05 1.02 2.24 1.79

TABLE 15 06RP68.HQ00009 (Jet Milled) + Plastiape RS00 Model 8 60 LPM 100LPM Mean % RSD Mean % RSD FPD (μg) 52.56 2.02 59.11 4.74 FPF (%) 33.970.86 36.01 4.20 MMAD (μm) 3.06 1.91 2.93 0.98 GSD 2.04 0.98 2.21 2.73

Based on these aerosol performance data, the wet polished drug particlesare preferred. They resulted in a higher fine particle dose, higher fineparticle fraction, a particle size distribution that would exhibitpenetration into both the central and peripheral lung regions, and wouldhave less oral deposition.

Example 10: Pharmacokinetic Modeling of Rapamycin

Based on the aerosol performance 06RP68.HQ00008 (Wet Polished)+PlasitapeRS01 Model as shown above, and the results of animal experiments inExample 3, it can be expected that delivery of inhaled rapamycindirectly to the lung in humans will similarly result in persistent lungconcentrations that are sufficiently high to be therapeuticallyeffective, but with low systemic exposure (low blood concentrations)thereby effectively minimizing side effects due to systemic exposure. Atwo compartment, pharmacokinetic model was developed to predict theconcentrations in the blood and lungs in humans after repeat QD dosingusing the formulation and DPI inhaler in Table 11. For thepharmacokinetic model, human PK parameters from the Rapamune® (NDA21-110, and NDA 21-083) summary basis of approval were used: the volumeof distribution was assumed to be 780 liters, clearance was0.0003/minute, and elimination half-life was 42.3 hours (assumingequivalency to rapamycin IV dosing). Absorption half-life of rapamycinfrom the lung was estimated to be approximately 0.5 hours, similar toother highly lipophilic compounds, such as fluticasone proprionate forwhich lung absorption data is available. Bioavailability of rapamycindepositing in the lung was assumed to be approximately 100%.Bioavailability of rapamycin absorbed by the GI route throughoropharyngeal deposition or removal from the upper airways bymucociliary clearance was assumed to be 14% as reported in the Rapamune®summary basis for approval. For a typical human inspiratory maneuver ata flow rate of 60 liters per minute, as shown in Table 11, the fineparticle dose was 57 micrograms, and the fine particle fraction was 40%.

The model predicts achieving an average steady state concentration after11 days as shown in FIG. 9. From the figure it can be seen that oncedaily repeat dosing of 57 micrograms delivered to the lungs results intrough blood concentrations of approximately 50 picograms/mL, andmaximum concentrations below 200 picograms/ml, substantially below theconcentrations of 5-15 ng/ml reported in McCormack et al. (2011),“Efficacy and safety of sirolimus in lymphangioleiomyomatosis”, N Engl JMed 364:1595-1606. Assuming a lung tissue mass of 850 grams, nometabolism in the lung and a lung absorption half life or 30 minutes, 57micrograms rapamycin delivered to the lungs would result in therapeuticlevels in the lung tissue, with local lung concentrations of rapamycinas high as approximately 60 ng/gram.

Example 11: Inhalation Toxicology Study in Rats and Dogs

The following study indicated that inhaled rapamycin is less toxic thanoral rapamycin.

In 28-day, repeat-dose, inhalation Good Laboratory Practice (GLP)toxicology studies with LAM-001 (inhaled rapamycin) in dogs and rats,toxicokinetic data (see Table 16) demonstrated that concentrations ofrapamycin were approximately 6 and 63-fold¹, respectively, higher thanthe systemic efficacious human blood concentrations found sufficient toinhibit mTOR activity in the lung of human LAM patients in the MILESstudy. In the most sensitive species, dogs exhibited a trough lung (24hour) to plasma ratio of approximately 16.7 at the NOAEL. ¹ Based onCmax on Day 28

TABLE 16 Results of animal studies. Lung/ Brain/ Lung/ Lung Brain BloodBlood Blood Brain Group 3 Day 28, 24-Hour Trough 32.2 2.4 1.9 16.7 1.213.7 6 animals at t = 24 Group 4 Day 28 24-Hour Trough 106.2 6.9 5.918.1 1.2 15.5 5 animals at t = 24 Group 5 Day 21, 24-Hour Trough 2273.224.4 23.4 97.3 1.0 0.6 3 animals at t = 24

Rats tolerated daily inhaled doses approximately 60 to 1700 times theintended therapeutic dose in humans. Dogs achieved a NOAEL 6 times theintended therapeutic dose in humans.

Administration of rapamycin via nose-only inhalation resulted in nochanges in respiratory function. The respiratory NOAELs of the achieveddoses established in the 28-day rat and dog studies were 8.91 mg/kg/dayand 0.0134 mg/kg/day, respectively; approximately 1713 and 6 times abovean orally inhaled recommended daily therapeutic dose (RDD) in humansbased on efficacy noted in the MILES trial. These data combined with theclinical data collected from the MILES trial indicate that a substantialbenefit in terms of safety may be achieved in patients administeredrapamycin via inhalation.

Furthermore, by delivering rapamycin via inhalation, first passmetabolism, oral absorption variability, and potential variability insystemic distribution to the lungs can be avoided. Physicians will beable to titrate the doses more efficiently and precisely, allowingpatients to receive efficacious lung doses while minimizing systemicexposure and the toxic effects of rapamycin. By varying the number ofcapsules in the dry powder inhaler, rapamycin will be able to deliverthe nominal efficacious dose thus a providing the patient a personalizedtreatment regimen which cannot be obtained with oral RAPAMUNE®.

It is anticipated that inhaled rapamycin will provide substantialclinical benefit over oral rapamycin (e.g., RAPAMUNE®) due to the directdelivery of the drug to the target tissue (lung) and the ability tocustomize treatment regimens.

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

What is claimed is:
 1. A method for the treatment of pulmonary arterialhypertension (PAH) in a human subject in need of such treatment, themethod comprising administering to the subject by inhalation apharmaceutical dry powder composition comprising micronized rapamycinparticles and particles of a carrier, wherein the micronized rapamycinparticles have a Mass Median Aerodynamic Diameter (MMAD) of from 2-3microns, wherein the composition is effective to deliver a therapeuticamount of the microparticulate rapamycin to the lungs of the humansubject, and wherein rapamycin is the only therapeutic agent in thecomposition.
 2. The method of claim 1, wherein the administering is oncedaily or twice daily by inhalation.
 3. The method of claim 1, whereinthe administering is performed using a dry powder delivery devicecomprising a reservoir containing a unit dosage form of thepharmaceutical dry powder composition.
 4. The method of claim 3, whereinthe unit dosage form contains from 50-150 micrograms of rapamycin. 5.The method of claim 3, wherein the unit dosage form is a capsulesuitable for use in the dry powder inhaler device.
 6. The method ofclaim 5, wherein the capsule is a gelatin, plastic, polymeric, orcellulosic capsule.
 7. The method of claim 3, wherein the unit dosageform is in the form of a foil/foil or foil/plastic blister.
 8. Themethod of claim 1, wherein the carrier is a saccharide or a sugaralcohol.
 9. The method of claim 1, wherein the carrier is selected fromthe group consisting of arabinose, glucose, fructose, ribose, mannose,sucrose, trehalose, lactose, maltose, a starch, dextran, mannitol,xylitol, and mixtures of any of the foregoing.
 10. The method of claim1, wherein the carrier comprises a blend of two different carriers, afirst carrier and a second carrier.
 11. The method of claim 10, whereinthe first carrier consists of particles having diameters of from 30-100microns and the second carrier consists of particles having diameters ofless than 10 microns.
 12. The method of claim 1, wherein the carrierdoes not comprise albumin.
 13. The method of claim 1, wherein thecarrier is not a carrier protein.
 14. The method of claim 1, wherein themicronized rapamycin particles are dispersed onto the surface ofparticles of the carrier.